Degradable clostridial toxins

ABSTRACT

The specification discloses modified Clostridial toxins comprising a PAR ligand domain, a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain and a Clostridial toxin binding domain; polynucleotide molecules encoding modified Clostridial toxins comprising a PAR ligand domain, a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain and a Clostridial toxin binding domain; and method of producing modified Clostridial toxins comprising a PAR ligand domain, a Clostridial toxin enzymatic domain, a Clostridial toxin translocation domain and a Clostridial toxin binding domain.

This is a continuation and claims priority pursuant to 35 U.S.C. §120 toU.S. patent application Ser. No. 12/192,905, filed Aug. 15, 2008, nowU.S. Pat. No. 7,______,______, which is a continuation of U.S. patentapplication Ser. No. 11/572,512, filed Jan. 23, 2007, now U.S. Pat. No.7,892,565, national stage application under 35 U.S.C. §371 of PCT patentapplication PCT/US2005/031613, filed on Sep. 1, 2005 which claims thebenefit of U.S. provisional patent application Ser. No. 10/931,719,which was filed on Jul. 5, 2005, which claims benefit to U.S.provisional patent application Ser. No. 60/651,494 filed Sep. 1, 2004,each of which is hereby incorporated by reference in its entirety.

All of the patents and publications cited in this application are herebyincorporated by reference in their entirety.

The ability of Clostridial toxins, such as, e.g., Botulinum neurotoxins(BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F and BoNT/G, andTetanus neurotoxin (TeNT), to inhibit neuronal transmission are beingexploited in a wide variety of therapeutic and cosmetic applications,see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OFBOTULINUM TOXIN (Slack, Inc., 2004). As an example, BOTOX® is currentlyapproved in one or more countries for the following indications:achalasia, adult spasticity, anal fissure, back pain, blepharospasm,bruxism, cervical dystonia, essential tremor, glabellar lines orhyperkinetic facial lines, headache, hemifacial spasm, hyperactivity ofbladder, hyperhidrosis, juvenile cerebral palsy, multiple sclerosis,myoclonic disorders, nasal labial lines, spasmodic dysphonia, strabismusand VII nerve disorder. In addition, Clostridial toxin therapies areproposed for treating neuromuscular disorders, see e.g., Kei Roger Aokiet al., Method for Treating Neuromuscular Disorders and Conditions withBotulinum Toxin Types A and B, U.S. Pat. No. 6,872,397 (Mar. 29, 2005);Rhett M. Schiffman, Methods for Treating Uterine Disorders, U.S. PatentPublication No. 2004/0175399 (Sep. 9, 2004); Richard L. Barron, Methodsfor Treating Ulcers and Gastroesophageal Reflux Disease, U.S. PatentPublication No. 2004/0086531 (May 7, 2004); and Kei Roger Aoki, et al.,Method for Treating Dystonia with Botulinum Toxin C to G, U.S. Pat. No.6,319,505 (Nov. 20, 2001); eye disorders, see e.g., Eric R. First,Methods and Compositions for Treating Eye Disorders, U.S. PatentPublication No. 2004/0234532 (Nov. 25, 2004); Kei Roger Aoki et al.,Botulinum Toxin Treatment for Blepharospasm, U.S. Patent Publication No.2004/0151740 (Aug. 5, 2004); and Kei Roger Aoki et al., Botulinum ToxinTreatment for Strabismus, U.S. Patent Publication No. 2004/0126396 (Jul.1, 2004); pain, see e.g., Kei Roger Aoki et al., Pain Treatment byPeripheral Administration of a Neurotoxin, U.S. Pat. No. 6,869,610 (Mar.22, 2005); Stephen Donovan, Clostridial Toxin Derivatives and Methods toTreat Pain, U.S. Pat. No. 6,641,820 (Nov. 4, 2003); Kei Roger Aoki, etal., Method for Treating Pain by Peripheral Administration of aNeurotoxin, U.S. Pat. No. 6,464,986 (Oct. 15, 2002); Kei Roger Aoki andMinglei Cui, Methods for Treating Pain, U.S. Pat. No. 6,113,915 (Sep. 5,2000); Martin A. Voet, Methods for Treating Fibromyalgia, U.S. Pat. No.6,623,742 (Sep. 23, 2003); Martin A. Voet, Botulinum Toxin Therapy forFibromyalgia, U.S. Patent Publication No. 2004/0062776 (Apr. 1, 2004);and Kei Roger Aoki et al., Botulinum Toxin Therapy for Lower Back Pain,U.S. Patent Publication No. 2004/0037852 (Feb. 26, 2004); muscleinjuries, see e.g., Gregory F. Brooks, Methods for Treating MuscleInjuries, U.S. Pat. No. 6,423,319 (Jul. 23, 2002); headache, see e.g.,Martin Voet, Methods for Treating Sinus Headache, U.S. Pat. No.6,838,434 (Jan. 4, 2005); Kei Roger Aoki et al., Methods for TreatingTension Headache, U.S. Pat. No. 6,776,992 (Aug. 17, 2004); and Kei RogerAoki et al., Method for Treating Headache, U.S. Pat. No. 6,458,365 (Oct.1, 2002); William J. Binder, Method for Reduction of Migraine HeadachePain, U.S. Pat. No. 5,714,469 (Feb. 3, 1998); cardiovascular diseases,see e.g., Gregory F. Brooks and Stephen Donovan, Methods for TreatingCardiovascular Diseases with Botulinum Toxin, U.S. Pat. No. 6,767,544(Jul. 27, 2004); neurological disorders, see e.g., Stephen Donovan,Parkinson's Disease Treatment, U.S. Pat. No. 6,620,415 (Sep. 16, 2003);and Stephen Donovan, Method for Treating Parkinson's Disease with aBotulinum Toxin, U.S. Pat. No. 6,306,403 (Oct. 23, 2001);neuropsychiatric disorders, see e.g., Stephen Donovan, Botulinum ToxinTherapy for Neuropsychiatric Disorders, U.S. Patent Publication No.2004/0180061 (Sep. 16, 2004); and Steven Donovan, Therapeutic Treatmentsfor Neuropsychiatric Disorders, U.S. Patent Publication No. 2003/0211121(Nov. 13, 2003); endocrine disorders, see e.g., Stephen Donovan, Methodfor Treating Endocrine Disorders, U.S. Pat. No. 6,827,931 (Dec. 7,2004); Stephen Donovan, Method for Treating Thyroid Disorders with aBotulinum Toxin, U.S. Pat. No. 6,740,321 (May 25, 2004); Kei Roger Aokiet al., Method for Treating a Cholinergic Influenced Sweat Gland, U.S.Pat. No. 6,683,049 (Jan. 27, 2004); Stephen Donovan, Neurotoxin Therapyfor Diabetes, U.S. Pat. No. 6,416,765 (Jul. 9, 2002); Stephen Donovan,Methods for Treating Diabetes, U.S. Pat. No. 6,337,075 (Jan. 8, 2002);Stephen Donovan, Method for Treating a Pancreatic Disorder with aNeurotoxin, U.S. Pat. No. 6,261,572 (Jul. 17, 2001); Stephen Donovan,Methods for Treating Pancreatic Disorders, U.S. Pat. No. 6,143,306 (Nov.7, 2000); cancers, see e.g., Stephen Donovan, Methods for Treating BoneTumors, U.S. Pat. No. 6,565,870 (May 20, 2003); Stephen Donovan, Methodfor Treating Cancer with a Neurotoxin to Improve Patient Function, U.S.Pat. No. 6,368,605 (Apr. 9, 2002); Stephen Donovan, Method for TreatingCancer with a Neurotoxin, U.S. Pat. No. 6,139,845 (Oct. 31, 2000); andMitchell F. Brin and Stephen Donovan, Methods for Treating DiverseCancers, U.S. Patent Publication No. 2005/0031648 (Feb. 10, 2005); oticdisorders, see e.g., Stephen Donovan, Neurotoxin Therapy for Inner EarDisorders, U.S. Pat. No. 6,358,926 (Mar. 19, 2002); and Stephen Donovan,Method for Treating Otic Disorders, U.S. Pat. No. 6,265,379 (Jul. 24,2001); autonomic disorders, see, e.g., Pankai J. Pasricha and Anthony N.Kalloo, Method for Treating Gastrointestinal Muscle Disorders and OtherSmooth Muscle Dysfunction, U.S. Pat. No. 5,437,291 (Aug. 1, 1995); aswell as other disorders, see e.g., William J. Binder, Method forTreatment of Skin Lesions Associated with Cutaneous Cell-proliferativeDisorders, U.S. Pat. No. 5,670,484 (Sep. 23, 1997); Eric R. First,Application of Botulinum Toxin to the Management of NeurogenicInflammatory Disorders, U.S. Pat. No. 6,063,768 (May 16, 2000); MarvinSchwartz and Brian J. Freund, Method to Reduce Hair Loss and StimulateHair Growth, U.S. Pat. No. 6,299,893 (Oct. 9, 2001); Jean D. A.Carruthers and Alastair Carruthers, Cosmetic Use of Botulinum Toxin forTreatment of Downturned Mouth, U.S. Pat. No. 6,358,917 (Mar. 19, 2002);Stephen Donovan, Use of a Clostridial Toxin to Reduce Appetite, U.S.Patent Publication No. 2004/40253274 (Dec. 16, 2004); and Howard I. Katzand Andrew M. Blumenfeld, Botulinum Toxin Dental Therapies andProcedures, U.S. Patent Publication No. 2004/0115139 (Jun. 17, 2004);Kei Roger Aoki, et al., Treatment of Neuromuscular Disorders andConditions with Different Botulinum, U.S. Patent Publication No.2002/0010138 (Jan. 24, 2002); and Kei Roger Aoki, et al., Use ofBotulinum Toxins for Treating Various Disorders and Conditions andAssociated Pain, U.S. Patent Publication No. 2004/0013692 (Jan. 22,2004). In addition, the expected use of Clostridial toxins, such as,e.g., BoNTs and TeNT, in therapeutic and cosmetic treatments of humansand other mammals is anticipated to expand to an ever widening range ofdiseases and ailments that can benefit from the properties of thesetoxins.

Clostridial toxin therapies are successfully used for many indications.Generally, administration of a Clostridial toxin is well tolerated.However, toxin administration in some applications can be challengingbecause of the larger doses required to achieve a beneficial effect.Larger doses can increase the likelihood that the toxin may move throughthe interstitial fluids and the circulatory systems, such as, e.g., thecardiovascular system and the lymphatic system, of the body, resultingin the undesirable dispersal of the toxin to areas not targeted fortoxin treatment. Such dispersal can lead to undesirable side effects,such as, e.g., inhibition of neurotransmitter release in neurons nottargeted for treatment or paralysis of a muscle not targeted fortreatment. For example, a patient administered a therapeuticallyeffective amount of a BoNT/A treatment into the neck muscles fortorticollis may develop dysphagia because of dispersal of the toxin intothe oropharynx. Thus, there remains a need for improved Clostridialtoxins that are effective at the site of treatment, but have negligibleto minimal effects in areas not targeted for a toxin treatment.

The growing clinical, therapeutic and cosmetic use of Clostridial toxinsin therapies requiring larger doses necessitates the pharmaceuticalindustry to develop modified Clostridial toxins that are effective atthe target site of the application, but reduce or prevent theundesirable side-effects associated with the dispersal of the toxins toan unwanted location or locations. The present invention provides novelClostridial toxins that reduce or prevent unwanted side-effectsassociated with toxin dispersal into non-targeted areas. These andrelated advantages are useful for various clinical, therapeutic andcosmetic applications, such as, e.g., the treatment of neuromusculardisorders, neuropathic disorders, eye disorders, pain, muscle injuries,headache, cardiovascular diseases, neuropsychiatric disorders, endocrinedisorders, cancers, otic disorders and hyperkinetic facial lines, aswell as, other disorders where a Clostridial toxin administration to amammal can produce a beneficial effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that activated PARs are predominantly targeted towardlysosomes for degradation. PARs are activated by an irreversiblemechanism, and once cleaved, most activated PARs are endocytosed anddirected, by intracellular trafficking routes, to lysosomes fordegradation. Step 1 illustrates cleavage of an inactivated PAR by aprotease to unmask the tethered ligand (black box). Step 2 illustratestethered ligand binding and conformational change of the activated PAR.Step 3 illustrates endocytosis of the activated PAR. Step 4 illustratesthe early and late endosomal sorting of the internalized activated PARthat result in the trafficking of the receptor to a lysosome. Step 5illustrates the degradation of the internalized activated PAR byproteases within the lysosome.

FIG. 2 shows modified Clostridial toxins comprising a tethered ligandare targeted toward lysosomes for degradation. Such modified toxins thatdiffuse into the circulatory system can bind to inactive PARs whichtriggers endocytosis and the directing of internalized toxins, byintracellular trafficking routes, to lysosomes for degradation. Step 1illustrates the binding of the modified Clostridial toxin comprising atethered ligand domain (black box) to a PAR. Step 2 illustratesendocytosis of the toxin-PAR complex. Step 3 illustrates the early andlate endosomal sorting of the internalized toxin-PAR complex that resultin the trafficking of the complex to a lysosome. Step 5 illustrates thedegradation of the internalized toxin-PAR complex by proteases withinthe lysosome.

FIG. 3 shows a schematic of the current paradigm of neurotransmitterrelease and Clostridial toxin intoxication in a central and peripheralneuron. FIG. 3 a shows a schematic for the neurotransmitter releasemechanism of a central and peripheral neuron. The release process can bedescribed as comprising two steps: 1) vesicle docking, where thevesicle-bound SNARE protein of a vesicle containing neurotransmittermolecules associates with the membrane-bound SNARE proteins located atthe plasma membrane; and 2) neurotransmitter release, where the vesiclefuses with the plasma membrane and the neurotransmitter molecules areexocytosed. FIG. 3 b shows a schematic of the intoxication mechanism fortetanus and botulinum toxin activity in a central and peripheral neuron.This intoxication process can be described as comprising four steps: 1)receptor binding, where a Clostridial toxin binds to a Clostridialreceptor system and initiates the intoxication process; 2) complexinternalization, where after toxin binding, a vesicle containing thetoxin/receptor system complex is endocytosed into the cell; 3) lightchain translocation, where multiple events are thought to occur,including, e.g., changes in the internal pH of the vesicle, formation ofa channel pore comprising the H_(N) domain of the Clostridial toxinheavy chain, separation of the Clostridial toxin light chain from theheavy chain, and release of the active light chain and 4) enzymatictarget modification, where the activate light chain of Clostridial toxinproteolytically cleaves its target SNARE substrate, such as, e.g.,SNAP-25, VAMP or Syntaxin, thereby preventing vesicle docking andneurotransmitter release.

FIG. 4 shows modified Clostridial toxins with a PAR ligand domainlocated at the amino terminus of the enzymatic domain. FIG. 4A depictsthe single polypeptide form of a Clostridial toxin with an amino tocarboxyl linear organization comprising a PAR ligand domain, anenzymatic domain, a translocation domain and a binding domain, with thedi-chain loop region depicted by the double SS bracket and the resultingdi-chain form after di-chain loop cleavage. In this example, a maskedPAR ligand domain is located at the amino terminus of the enzymaticdomain and a proteolytic cleavage site (P1) is located in front of thePAR ligand binding domain. Upon proteolytic cleavage with a P1 protease,the PAR ligand domain becomes unmasked. P2 is a protease cleavage siteused to convert the single chain form of the toxin to the di-chain form.Both P1 and P2 can be a PAR endogenous protease cleavage site or anexogenous protease cleavage site and can be cleaved by the same proteaseor different proteases. FIG. 4B depicts the single polypeptide form of aClostridial toxin with an amino to carboxyl linear organizationcomprising a PAR ligand domain, an enzymatic domain, a binding domainand a translocation domain, with the di-chain loop region depicted bythe double SS bracket. In this example, a masked PAR ligand domain islocated at the amino terminus of the enzymatic domain and a proteolyticcleavage site (P1) is located in front of the PAR ligand binding domain.Upon proteolytic cleavage with a P1 protease, the PAR ligand domainbecomes unmasked. P2 is a protease cleavage site used to convert thesingle chain form of the toxin to the di-chain form. Both P1 and P2 canbe a PAR endogenous protease cleavage site or an exogenous proteasecleavage site and can be cleaved by the same protease or differentproteases. FIG. 4C depicts the single polypeptide form of a Clostridialtoxin with an amino to carboxyl linear organization comprising a PARligand domain, an enzymatic domain, a translocation domain and a bindingdomain, with the di-chain loop region depicted by the double SS bracket.In this example, an unmasked PAR ligand domain is located at the aminoterminus of the enzymatic domain. P2 is a protease cleavage site used toconvert the single chain form of the toxin to the di-chain form and canbe a PAR endogenous protease cleavage site or an exogenous proteasecleavage site. FIG. 4D depicts the single polypeptide form of aClostridial toxin with an amino to carboxyl linear organizationcomprising a PAR ligand domain, an enzymatic domain, a binding domainand a translocation domain, with the di-chain loop region depicted bythe double SS bracket. In this example, an unmasked PAR ligand domain islocated at the amino terminus of the enzymatic domain. P2 is a proteasecleavage site used to convert the single chain form of the toxin to thedi-chain form and can be a PAR endogenous protease cleavage site or anexogenous protease cleavage site.

FIG. 5 shows modified Clostridial toxins with a PAR ligand domainlocated at the amino terminus of the translocation domain. FIG. 5Adepicts the single polypeptide form of a Clostridial toxin with an aminoto carboxyl linear organization comprising a binding domain, anenzymatic domain, a PAR ligand domain and a translocation domain, withthe di-chain loop region depicted by the double SS bracket and theresulting di-chain form after di-chain loop cleavage. In this example, amasked PAR ligand domain is located at the amino terminus of thetranslocation domain and a proteolytic cleavage site (P1) is located infront of the PAR ligand binding domain. Upon proteolytic cleavage with aP1 protease, the PAR ligand domain becomes unmasked. P1 is also theprotease cleavage site used to convert the single chain form of thetoxin to the di-chain form. P1 can be a PAR endogenous protease cleavagesite or an exogenous protease cleavage site. FIG. 5B depicts the singlepolypeptide form of a Clostridial toxin with an amino to carboxyl linearorganization comprising an enzymatic domain, a PAR ligand domain, atranslocation domain and a binding domain, with the di-chain loop regiondepicted by the double SS bracket. In this example, a masked PAR liganddomain is located at the amino terminus of the translocation domain anda proteolytic cleavage site (P1) is located in front of the PAR ligandbinding domain. Upon proteolytic cleavage with a P1 protease, the PARligand domain becomes unmasked. P1 is also the protease cleavage siteused to convert the single chain form of the toxin to the di-chain form.P1 can be a PAR endogenous protease cleavage site or an exogenousprotease cleavage site.

FIG. 6 shows modified Clostridial toxins with a PAR ligand domainlocated at the amino terminus of the binding domain. FIG. 6 depicts thesingle polypeptide form of a Clostridial toxin with an amino to carboxyllinear organization comprising an enzymatic domain, a PAR ligand domain,a binding domain and a translocation domain, with the di-chain loopregion depicted by the double SS bracket and the resulting di-chain formafter di-chain loop cleavage. In this example, a masked PAR liganddomain is located at the amino terminus of the binding domain and aproteolytic cleavage site (P1) is located in front of the PAR ligandbinding domain. Upon proteolytic cleavage with a P1 protease, the PARligand domain becomes unmasked. P1 is also the protease cleavage siteused to convert the single chain form of the toxin to the di-chain form.P1 can be a PAR endogenous protease cleavage site or an exogenousprotease cleavage site.

FIG. 7 shows a plasmid map of prokaryotic expression constructpET29b/BoNT/A-ED-PAR1Tb comprising a polynucleotide molecule of SEQ IDNO: 136 encoding a modified BoNT/A of SEQ ID NO: 85, operably-linked toa carboxyl-terminal polyhistidine binding polypeptide. A Trypsinprotease cleavage site is operably-linked between the polyhistidinebinding polypeptide and the modified BoNT/A. Abbreviations are asfollows: P_(T7), a bacteriophage T7 promoter region; Thrombin, apolynucleotide molecule encoding a PAR1 Thrombin cleavage site; PAR1-LD,a polynucleotide molecule encoding a PAR1 ligand domain; ED, apolynucleotide molecule encoding a BoNT/A enzymatic domain; TD, apolynucleotide molecule encoding a BoNT/A translocation domain; BD, apolynucleotide molecule encoding a BoNT/A binding domain; Trypsin, apolynucleotide molecule encoding Trypsin cleavage site; 6×His, apolynucleotide molecule encoding a polyhistidine binding polypeptide; T7TT, a bacteriophage T7 transcription termination region; f1 origin, abacteriophage f1 origin of replication; Kanamycin, a polynucleotidemolecule encoding an aminophosphotransferase that confers Kanamycinresistance; pBR322 ori, a pBR322 origin of plasmid replication region;lacI, a polynucleotide molecule encoding a lactose I.

FIG. 8 shows a plasmid map of prokaryotic expression constructpET29b/BoNT/A-TD-PAR1Tb comprising a polynucleotide molecule of SEQ IDNO: 144 encoding a modified BoNT/A of SEQ ID NO: 93, operably-linked toa carboxyl-terminal polyhistidine binding polypeptide. A Trypsinprotease cleavage site is operably-linked between the polyhistidinebinding polypeptide and the modified BoNT/A. Abbreviations are asfollows: P_(T7), a bacteriophage T7 promoter region; ED, apolynucleotide molecule encoding a BoNT/A enzymatic domain; Thrombin, apolynucleotide molecule encoding a PAR1 Thrombin cleavage site; PAR1-LD,a polynucleotide molecule encoding a PAR1 ligand domain; TD, apolynucleotide molecule encoding a BoNT/A translocation domain; BD, apolynucleotide molecule encoding a BoNT/A binding domain; Trypsin, apolynucleotide molecule encoding Trypsin cleavage site; 6×His, apolynucleotide molecule encoding a polyhistidine binding polypeptide; T7TT, a bacteriophage T7 transcription termination region; f1 origin, abacteriophage f1 origin of replication; Kanamycin, a polynucleotidemolecule encoding an aminophosphotransferase that confers Kanamycinresistance; pBR322 ori, a pBR322 origin of plasmid replication region;lacI, a polynucleotide molecule encoding a lactose I.

FIG. 9 shows a plasmid map of prokaryotic expression constructpET29b/BoNT/A-BD-PAR1Tb comprising a polynucleotide molecule of SEQ IDNO: 152 encoding a modified BoNT/A of SEQ ID NO: 101, operably-linked toa carboxyl-terminal polyhistidine binding polypeptide. A Trypsinprotease cleavage site is operably-linked between the polyhistidinebinding polypeptide and the modified BoNT/A. Abbreviations are asfollows: P_(T7), a bacteriophage T7 promoter region; Thrombin, apolynucleotide molecule encoding a PAR1 Thrombin cleavage site; PAR1-LD,a polynucleotide molecule encoding a PAR1 ligand domain; ED, apolynucleotide molecule encoding a BoNT/A enzymatic domain; TD, apolynucleotide molecule encoding a BoNT/A translocation domain; BD, apolynucleotide molecule encoding a BoNT/A binding domain; Trypsin, apolynucleotide molecule encoding Trypsin cleavage site; 6×His, apolynucleotide molecule encoding a polyhistidine binding polypeptide; T7TT, a bacteriophage T7 transcription termination region; f1 origin, abacteriophage f1 origin of replication; Kanamycin, a polynucleotidemolecule encoding an aminophosphotransferase that confers Kanamycinresistance; pBR322 ori, a pBR322 origin of plasmid replication region;lacI, a polynucleotide molecule encoding a lactose I.

FIG. 10 shows a plasmid map of yeast expression construct pPICZA/BoNT/A-ED-PAR1Tb comprising a polynucleotide molecule of SEQ ID NO:136 encoding a modified BoNT/A of SEQ ID NO: 85, operably-linked tocarboxyl-terminal c-myc and polyhistidine binding polypeptides.Abbreviations are as follows: P_(AOX1), an aldehyde oxidase 1 promoterregion; Thrombin, a polynucleotide molecule encoding a PAR1 Thrombincleavage site; PAR1-LD, a polynucleotide molecule encoding a PAR1 liganddomain; ED, a polynucleotide molecule encoding a BoNT/A enzymaticdomain; TD, a polynucleotide molecule encoding a BoNT/A translocationdomain; BD, a polynucleotide molecule encoding a BoNT/A binding domain;c-myc, a polynucleotide molecule encoding a c-myc binding polypeptide;6×His, a polynucleotide molecule encoding a polyhistidine bindingpolypeptide; AOX1 TT, an aldehyde oxidase 1 transcription terminationregion; Zeocin™, a polynucleotide molecule encoding a Zeocin™ resistancepolypeptide; pUC ori, a pUC origin of plasmid replication region.

FIG. 11 shows a plasmid map of baculovirus transfer constructpBACgus3/BoNT/A-ED-PAR1Tb comprising a polynucleotide molecule of SEQ IDNO: 136 encoding a modified BoNT/A of SEQ ID NO: 85, operably-linked tocarboxyl-terminal polyhistidine binding polypeptide. A Thrombin proteasecleavage site is operably-linked between the modified BoNT/A and thepolyhistidine binding polypeptide. Abbreviations are as follows: P_(PH),an polyhedrin promoter region; gp64, a polynucleotide molecule encodinga gp64 signal polypeptide; Thrombin, a polynucleotide molecule encodinga PAR1 Thrombin cleavage site; PAR1-LD, a polynucleotide moleculeencoding a PAR1 ligand domain; ED, a polynucleotide molecule encoding aBoNT/A enzymatic domain; TD, a polynucleotide molecule encoding a BoNT/Atranslocation domain; BD, a polynucleotide molecule encoding a BoNT/Abinding domain; Thrombin, a polynucleotide molecule encoding a Thrombinprotease cleavage site; 6×His, a polynucleotide molecule encoding apolyhistidine binding polypeptide; pUC ori, a pUC origin of plasmidreplication region; Ampicillin, a polynucleotide molecule encoding aβ-lactamase that confers Ampicillin resistance; f1 ori, a bacteriophagef1 origin of replication; gus, a polynucleotide molecule encoding aβ-glucuronidase.

FIG. 12 shows a plasmid map of mammalian expression constructpSecTag2/BoNT/A-ED-PAR1Tb comprising a polynucleotide molecule of SEQ IDNO: 136 encoding a modified BoNT/A of SEQ ID NO: 85, operably-linked tocarboxyl-terminal c-myc and polyhistidine binding polypeptides.Abbreviations are as follows: P_(CMV), an cytomegalovirus promoterregion; IgK, a polynucleotide molecule encoding an immunoglobulin Kpolypeptide; Thrombin, a polynucleotide molecule encoding a PAR1Thrombin cleavage site; PAR1-LD, a polynucleotide molecule encoding aPAR1 ligand domain; ED, a polynucleotide molecule encoding a BoNT/Aenzymatic domain; TD, a polynucleotide molecule encoding a BoNT/Atranslocation domain; BD, a polynucleotide molecule encoding a BoNT/Abinding domain; c-myc, a polynucleotide molecule encoding a c-mycbinding polypeptide; 6×His, a polynucleotide molecule encoding apolyhistidine binding polypeptide; BGH pA, a bovine growth hormonepolyadenylation site; f1 ori, a bacteriophage f1 origin of replication;P_(SV40), a simian virus 40 promoter region; Zeocin™, a region encodingan Zeocin™ resistance polypeptide; pUC ori, a pUC origin of plasmidreplication region; Ampicillin, a polynucleotide molecule encoding aβ-lactamase that confers Ampicillin resistance.

DETAILED DESCRIPTION

While all details of this process are not yet precisely known,protease-activated G protein-coupled receptor (PAR) signaling elicitsresponses according to the classic paradigm established for other GPCRs.Although the applicants have no wish to be limited by the followingdescription, the overall signaling mechanism can be described ascomprising at least four steps: 1) receptor activation where theprotease agonist cleaves a specific site located at the extracellularamino-terminus of the receptor that generates a new amino acid terminusthat that functions as a tethered ligand; 2) ligand binding where theunmasked tethered ligand binds to the ligand binding domain located inthe second extracellular loop of the receptor resulting in aconformational change of the cleaved PAR that promotes intracellularinteractions with heteromeric G proteins; 3) signal transduction where,in common with most GPCRs, the PAR-G protein complex signals throughvarious Gq-, Gi- and Gβγ-mediated signaling pathways in a temporal andspatial manner; and 4) signal termination where receptor desensitizationand receptor degradation stop the signaling of the activated complex(FIG. 1), see, e.g., Joann Trejo, Protease-Activated Receptors NewConcepts in Regulation of G Protein-Coupled Receptor Signaling andTrafficking, 307 (2) J. Pharmacol. Exp. Ther. 437-442 (2003); andValeria S. Ossovskaya and Nigel W. Bennett, Protease-ActivatedReceptors: Contribution to Physiology and Disease, 84 (2) Physiol. Rev.579-621 (2004).

Despite the irreversible mechanism of receptor activation, signalinginitiated by activated PARs appears to be rapidly and efficientlyterminated. Signal termination is especially important for regulatingthe magnitude, duration and fidelity of PAR-elicited cellular responsesand appears to be governed by two processes. The first mechanism isreceptor desensitization, where enzymatic phosphorylation of theactivated PAR by G-protein Receptor Kinases (GRKs) and other kinasesuncouple the activated receptor from its associated G proteins andsignaling effectors. The second mechanism of PAR-initiated signaltermination is receptor degradation, where proteolytic cleavage of theactivated PAR by cell-surface proteases on the plasma membrane and byintracellular proteases within lysosomal vesicles destroys the activatedreceptors. Because of the irreversible nature of PAR activation,internalization of activated PARs and their subsequent sorting tolysosomes appears to be the dominant process for signal termination.Internalization of activated PARs contributes to signal termination bothby removing activated receptors from G proteins and signaling effectorsand by directing activated receptors to lysosomal vesicles whereproteolytic degradation effectively inactivates the activated receptor.In addition to endocytosis of activated receptors, PARs also undergoconstitutive endocytosis in the absence of proteolytic activation.Therefore, the unusual and irreversible mode of PAR activation has givenrise to a very rapid and efficient means of terminating the signalingevents elicited by activated PARs utilizing endocytosis and lysosomaldegradation.

The present invention discloses modified Clostridial toxins that can berapidly removed from the circulatory system by exploiting the processesinvolved in activated PAR signal termination. Clostridial toxinscontaining a PAR ligand domain can bind PARs, which initiates theinternalization and degradation of such modified toxins. Many tissues ofthe cardiovascular system and lymphatic system comprise cells whichexpress PARs. In situations where a modified Clostridial toxincomprising a PAR ligand domain has diffused into a circulatory system,this modified toxin can be effectively internalized by a PAR expressingcell and degraded by proteases within lysosomes (FIG. 2). Thus utilizingthe processes involved in PAR-elicited signal termination will lessen orremove a Clostridial toxin from the circulatory system thereby reducingor preventing the undesirable side-effects associated with the diffusionof a Clostridial toxin to an unwanted location.

Aspects of the present invention provide modified Clostridial toxinscomprising a PAR ligand domain; a Clostridial toxin enzymatic domain; aClostridial toxin translocation domain; and a Clostridial toxin bindingdomain. It is envisioned that the location of the PAR ligand domain inthe modified Clostridial toxins of the present specification is locatedat a free amino terminus, including, without limitation, at the aminoterminus of the Clostridial toxin enzymatic domain; at the aminoterminus of the Clostridial toxin translocation domain; and at the aminoterminus of the Clostridial toxin binding domain. Thus, in embodiments,the modified Clostridial toxins comprise a PAR ligand domain; aClostridial toxin enzymatic domain; a Clostridial toxin translocationdomain; and a Clostridial toxin binding domain; wherein the PAR liganddomain is located at the amino terminus of the Clostridial toxinenzymatic domain. In other embodiments, the modified Clostridial toxinscomprise a PAR ligand domain; a Clostridial toxin enzymatic domain; aClostridial toxin translocation domain; and a Clostridial toxin bindingdomain; wherein the PAR ligand domain is located at the amino terminusof the Clostridial toxin translocation domain. In still otherembodiments, the modified Clostridial toxins comprise a PAR liganddomain; a Clostridial toxin enzymatic domain; a Clostridial toxintranslocation domain; and a Clostridial toxin binding domain; whereinthe PAR ligand domain is located at the amino terminus of theClostridial toxin binding domain.

Other aspects of the present invention provide polynucleotide moleculesencoding modified Clostridial toxins comprising a PAR ligand domain; aClostridial toxin enzymatic domain; a Clostridial toxin translocationdomain; and a Clostridial toxin binding domain. It is envisioned thatthe location of the PAR ligand domain of the modified Clostridial toxinsencoded by polynucleotide molecules of the present specification islocated at a free amino terminus, including, without limitation, at theamino terminus of the Clostridial toxin enzymatic domain; at the aminoterminus of the Clostridial toxin translocation domain; and at the aminoterminus of the Clostridial toxin binding domain. Thus, in embodiments,the polynucleotide molecules encoded modified Clostridial toxinscomprising a PAR ligand domain; a Clostridial toxin enzymatic domain; aClostridial toxin translocation domain; and a Clostridial toxin bindingdomain; wherein the PAR ligand domain is located at the amino terminusof the Clostridial toxin enzymatic domain. In other embodiments, thepolynucleotide molecules encoded modified Clostridial toxins comprisinga PAR ligand domain; a Clostridial toxin enzymatic domain; a Clostridialtoxin translocation domain; and a Clostridial toxin binding domain;wherein the PAR ligand domain is located at the amino terminus of theClostridial toxin translocation domain. In still other embodiments, thepolynucleotide molecules encoded modified Clostridial toxins comprisinga PAR ligand domain; a Clostridial toxin enzymatic domain; a Clostridialtoxin translocation domain; and a Clostridial toxin binding domain;wherein the PAR ligand domain is located at the amino terminus of theClostridial toxin binding domain.

Other aspects of the present invention provide methods of producing amodified Clostridial toxin comprising a PAR ligand domain; a Clostridialtoxin enzymatic domain; a Clostridial toxin translocation domain; and aClostridial toxin binding domain, such method comprising the step ofexpressing in a cell a polynucleotide molecule encoding a modifiedClostridial toxin comprising a PAR ligand domain; a Clostridial toxinenzymatic domain; a Clostridial toxin translocation domain; and aClostridial toxin binding domain. Other aspects of the present inventionprovide methods of producing in a cell a modified Clostridial toxincomprising a PAR ligand domain; a Clostridial toxin enzymatic domain; aClostridial toxin translocation domain; and a Clostridial toxin bindingdomain, such method comprising the steps of introducing in a cell anexpression construct comprising a polynucleotide molecule encoding amodified Clostridial toxin comprising a PAR ligand domain; a Clostridialtoxin enzymatic domain; a Clostridial toxin translocation domain; and aClostridial toxin binding domain and expressing the expression constructin the cell.

Aspects of the present invention provide, in part, a Clostridial toxin.As used herein, the term “Clostridial toxin” means any polypeptide thatcan execute the overall cellular mechanism whereby a Clostridial toxinenters a neuron and inhibits neurotransmitter release and encompassesthe binding of a Clostridial toxin to a low or high affinity receptorcomplex, the internalization of the toxin/receptor complex, thetranslocation of the Clostridial toxin light chain into the cytoplasmand the enzymatic modification of a Clostridial toxin substrate.Clostridia toxins produced by Clostridium botulinum, Clostridium tetani,Clostridium baratii and Clostridium butyricum are the most widely usedin therapeutic and cosmetic treatments of humans and other mammals.Strains of C. botulinum produce seven antigenically-distinct types ofBotulinum toxins (BoNTs), which have been identified by investigatingbotulism outbreaks in man (BoNT/A, /B, /E and /F), animals (BoNT/C1 and/D), or isolated from soil (BoNT/G). BoNTs possess approximately 35%amino acid identity with each other and share the same functional domainorganization and overall structural architecture. It is recognized bythose of skill in the art that within each type of Clostridial toxinthere can be subtypes that differ somewhat in their amino acid sequence,and also in the nucleic acids encoding these proteins. For example,there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 andBoNT/A4, with specific subtypes showing approximately 89% amino acididentity when compared to another BoNT/A subtype. While all seven BoNTserotypes have similar structure and pharmacological properties, eachalso displays heterogeneous bacteriological characteristics. Incontrast, tetanus toxin (TeNT) is produced by a uniform group of C.tetani. Two other species of Clostridia, C. baratii and C. butyricum,also produce toxins similar to BoNT/F and BoNT/E, respectively.

Clostridial toxins are each translated as a single chain polypeptide ofapproximately 150 kDa that is subsequently cleaved by proteolyticscission within a disulfide loop by a naturally-occurring protease, suchas, e.g., an endogenous Clostridial toxin protease or anaturally-occurring proteases produced in the environment. Thisposttranslational processing yields a di-chain molecule comprising anapproximately 50 kDa light chain (LC) and an approximately 100 kDa heavychain (HC) held together by a single disulfide bond and noncovalentinteractions. Each mature di-chain molecule comprises three functionallydistinct domains: 1) an enzymatic domain located in the LC that includesa metalloprotease region containing a zinc-dependent endopeptidaseactivity which specifically targets core components of theneurotransmitter release apparatus (Table 1); 2) a translocation domaincontained within the amino-terminal half of the HC (H_(N)) thatfacilitates release of the LC from intracellular vesicles into thecytoplasm of the target cell (Table 1); and 3) a binding domain foundwithin the carboxyl-terminal half of the HC (H_(C)) that determines thebinding activity and binding specificity of the toxin to the receptorcomplex located at the surface of the target cell (Table 1).

The binding, translocation and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby Clostridial toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of type.Although the applicants have no wish to be limited by the followingdescription, the intoxication mechanism can be described as comprisingat least four steps: 1) receptor binding, 2) complex internalization, 3)light chain translocation, and 4) enzymatic target modification (seeFIG. 1). The process is initiated when the H_(C) domain of a Clostridialtoxin binds to a toxin-specific receptor complex located on the plasmamembrane surface of a target cell. The binding specificity of a receptorcomplex is thought to be achieved, in part, by specific combinations ofgangliosides and protein receptors that appear to distinctly compriseeach Clostridial toxin receptor complex. Once bound, the toxin/receptorcomplexes are internalized by endocytosis and the internalized vesiclesare sorted to specific intracellular routes. The translocation stepappears to be triggered by the acidification of the vesicle compartment.This process seems to initiate two important pH-dependent structuralrearrangements that increase hydrophobicity and promote formationdi-chain form of the toxin. Once activated, light chain endopeptidase ofthe toxin is released from the intracellular vesicle into the cytosolwhere it specifically targets one of three known core components of theneurotransmitter release apparatus. These core proteins,vesicle-associated membrane protein (VAMP)/synaptobrevin,synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, arenecessary for synaptic vesicle docking and fusion at the nerve terminaland constitute members of the soluble N-ethylmaleimide-sensitivefactor-attachment protein-receptor (SNARE) family. BoNT/A and BoNT/Ecleave SNAP-25 in the carboxyl-terminal region, releasing a nine ortwenty-six amino acid segment, respectively, and BoNT/C1 also cleavesSNAP-25 near the carboxyl-terminus. The botulinum serotypes BoNT/B,BoNT/D, BoNT/F and BoNT/G, and tetanus toxin, act on the conservedcentral portion of VAMP, and release the amino-terminal portion of VAMPinto the cytosol. BoNT/C1 cleaves syntaxin at a single site near thecytosolic membrane surface. The selective proteolysis of synaptic SNAREsaccounts for the block of neurotransmitter release caused by Clostridialtoxins in vivo. The SNARE protein targets of Clostridial toxins arecommon to exocytosis in a variety of non-neuronal types; in these cells,as in neurons, light chain peptidase activity inhibits exocytosis, see,e.g., Yann Humeau et al., How Botulinum and Tetanus Neurotoxins BlockNeurotransmitter Release, 82 (5) Biochimie. 427-446 (2000); KathrynTurton et al., Botulinum and Tetanus Neurotoxins: Structure, Functionand Therapeutic Utility, 27 (11) Trends Biochem. Sci. 552-558. (2002);Giovanna Lalli et al., The Journey of Tetanus and Botulinum Neurotoxinsin Neurons, 11 (9) Trends Microbiol. 431-437, (2003).

TABLE 1 Clostridial Toxin Reference Sequences and Regions Toxin SEQ IDNO: LC H_(N) H_(C) BoNT/A 1 M1-K448 A449-K871 N872-L1296 BoNT/B 2M1-K441 A442-S858 E859-E1291 BoNT/C1 3 M1-K449 T450-N866 N867-E1291BoNT/D 4 M1-R445 D446-N862 S863-E1276 BoNT/E 5 M1-R422 K423-K845R846-K1252 BoNT/F 6 M1-K439 A440-K864 K865-E1274 BoNT/G 7 M1-K446S447-S863 N864-E1297 TeNT 8 M1-A457 S458-V879 I880-D1315

A Clostridial toxin includes, without limitation, naturally occurringClostridial toxin variants, such as, e.g., Clostridial toxin isoformsand Clostridial toxin subtypes; non-naturally occurring Clostridialtoxin variants, such as, e.g., conservative Clostridial toxin variants,non-conservative Clostridial toxin variants, Clostridial toxin chimericvariants and active Clostridial toxin fragments thereof, or anycombination thereof. As used herein, the term “Clostridial toxinvariant,” whether naturally-occurring or non-naturally-occurring, meansa Clostridial toxin that has at least one amino acid change from thecorresponding region of the disclosed reference sequences (see Table 1)and can be described in percent identity to the corresponding region ofthat reference sequence. As non-limiting examples, a BoNT/A variantcomprising amino acids 1-1296 of SEQ ID NO: 1 will have at least oneamino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1-1296 of SEQID NO: 1; a BoNT/B variant comprising amino acids 1-1291 of SEQ ID NO: 2will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-1291 of SEQ ID NO: 2; a BoNT/C1 variant comprising amino acids1-1291 of SEQ ID NO: 3 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 1-1291 of SEQ ID NO: 3; a BoNT/Dvariant comprising amino acids 1-1276 of SEQ ID NO: 4 will have at leastone amino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1-1276 of SEQID NO: 4; a BoNT/E variant comprising amino acids 1-1252 of SEQ ID NO: 5will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-1252 of SEQ ID NO: 5; a BoNT/F variant comprising amino acids1-1274 of SEQ ID NO: 6 will have at least one amino acid difference,such as, e.g., an amino acid substitution, deletion or addition, ascompared to the amino acid region 1-1274 of SEQ ID NO: 6; a BoNT/Gvariant comprising amino acids 1-1297 of SEQ ID NO: 7 will have at leastone amino acid difference, such as, e.g., an amino acid substitution,deletion or addition, as compared to the amino acid region 1-1297 of SEQID NO: 7; and a TeNT variant comprising amino acids 1-1315 of SEQ ID NO:8 will have at least one amino acid difference, such as, e.g., an aminoacid substitution, deletion or addition, as compared to the amino acidregion 1-1315 of SEQ ID NO: 8.

Any of a variety of sequence alignment methods can be used to determinepercent identity, including, without limitation, global methods, localmethods and hybrid methods, such as, e.g., segment approach methods.Protocols to determine percent identity are routine procedures withinthe scope of one skilled in the art and from the teaching herein.

Global methods align sequences from the beginning to the end of themolecule and determine the best alignment by adding up scores ofindividual residue pairs and by imposing gap penalties. Non-limitingmethods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al.,CLUSTAL W: Improving the Sensitivity of Progressive Multiple SequenceAlignment Through Sequence Weighting, Position-Specific Gap Penaltiesand Weight Matrix Choice, 22 (22) Nucleic Acids Research 4673-4680(1994); and iterative refinement, see, e.g., Osamu Gotoh, SignificantImprovement in Accuracy of Multiple Protein Sequence Alignments byIterative Refinement as Assessed by Reference to Structural Alignments,264 (4) J. Mol. Biol. 823-838 (1996).

Local methods align sequences by identifying one or more conservedmotifs shared by all of the input sequences. Non-limiting methodsinclude, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans,Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignmentof Several Protein Sequences, 8 (5) CABIOS 501-509 (1992); Gibbssampling, see, e.g., C. E. Lawrence et al., Detecting Subtle SequenceSignals: A Gibbs Sampling Strategy for Multiple Alignment, 262 (5131)Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al.,Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20 (9) Bioinformatics,:1428-1435 (2004).

Hybrid methods combine functional aspects of both global and localalignment methods. Non-limiting methods include, e.g.,segment-to-segment comparison, see, e.g., Burkhard Morgenstern et al.,Multiple DNA and Protein Sequence Alignment Based On Segment-To-SegmentComparison, 93 (22) Proc. Natl. Acad. Sci. U.S.A. 12098-12103 (1996);T-Coffee, see, e.g., Cédric Notredame et al., T-Coffee: A NovelAlgorithm for Multiple Sequence Alignment, 302 (1) J. Mol. Biol. 205-217(2000); MUSCLE, see, e.g., Robert C. Edgar, MUSCLE: Multiple SequenceAlignment With High Score Accuracy and High Throughput, 32 (5) NucleicAcids Res. 1792-1797 (2004); and DIALIGN-T, see, e.g., Amarendran RSubramanian et al., DIALIGN-T: An Improved Algorithm for Segment-BasedMultiple Sequence Alignment, 6 (1) BMC Bioinformatics 66 (2005).

As used herein, the term “naturally occurring Clostridial toxin variant”means any Clostridial toxin produced without the aid of any humanmanipulation, including, without limitation, Clostridial toxin isoformsproduced from alternatively-spliced transcripts, Clostridial toxinisoforms produced by spontaneous mutation and Clostridial toxinsubtypes. Non-limiting examples of a Clostridial toxin isoform include,e.g., BoNT/A isoforms, BoNT/B isoforms, BoNT/C1 isoforms, BoNT/Disoforms, BoNT/E isoforms, BoNT/F isoforms, BoNT/G isoforms, and TeNTisoforms. Non-limiting examples of a Clostridial toxin subtype include,e.g., BoNT/A subtypes BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4; BoNT/Bsubtypes BoNT/B1, BoNT/B2, BoNT/B bivalent and BoNT/B nonproteolytic;BoNT/C1 subtypes BoNT/C1-1 and BoNT/C1-2; BoNT/E subtypes BoNT/E1,BoNT/E2 and BoNT/E3; and BoNT/F subtypes BoNT/F1, BoNT/F2, BoNT/F3 andBoNT/F4.

As used herein, the term “non-naturally occurring Clostridial toxinvariant” means any Clostridial toxin produced with the aid of humanmanipulation, including, without limitation, Clostridial toxins producedby genetic engineering using random mutagenesis or rational design andClostridial toxins produced by chemical synthesis. Non-limiting examplesof non-naturally occurring Clostridial toxin variants include, e.g.,conservative Clostridial toxin variants, non-conservative Clostridialtoxin variants, Clostridial toxin chimeric variants and activeClostridial toxin fragments.

As used herein, the term “conservative Clostridial toxin variant” meansa Clostridial toxin that has at least one amino acid substituted byanother amino acid or an amino acid analog that has at least oneproperty similar to that of the original amino acid from the referenceClostridial toxin sequence (Table 1). Examples of properties include,without limitation, similar size, topography, charge, hydrophobicity,hydrophilicity, lipophilicity, covalent-bonding capacity,hydrogen-bonding capacity, a physicochemical property, of the like, orany combination thereof. A conservative Clostridial toxin variant canfunction in substantially the same manner as the reference Clostridialtoxin on which the conservative Clostridial toxin variant is based, andcan be substituted for the reference Clostridial toxin in any aspect ofthe present invention. A conservative Clostridial toxin variant maysubstitute one or more amino acids, two or more amino acids, three ormore amino acids, four or more amino acids, five or more amino acids,ten or more amino acids, 20 or more amino acids, 30 or more amino acids,40 or more amino acids, 50 or more amino acids, 100 or more amino acids,200 or more amino acids, 300 or more amino acids, 400 or more aminoacids, or 500 or more amino acids from the reference Clostridial toxinon which the conservative Clostridial toxin variant is based. Aconservative Clostridial toxin variant can also substitute at least 10contiguous amino acids, at least 15 contiguous amino acids, at least 20contiguous amino acids, or at least 25 contiguous amino acids from thereference Clostridial toxin on which the conservative Clostridial toxinvariant is based, that possess at least 50% amino acid identity, 65%amino acid identity, 75% amino acid identity, 85% amino acid identity or95% amino acid identity to the reference Clostridial toxin on which theconservative Clostridial toxin variant is based. Non-limiting examplesof a conservative Clostridial toxin variant include, e.g., conservativeBoNT/A variants, conservative BoNT/B variants, conservative BoNT/C1variants, conservative BoNT/D variants, conservative BoNT/E variants,conservative BoNT/F variants, conservative BoNT/G variants, andconservative TeNT variants.

As used herein, the term “non-conservative Clostridial toxin variant”means a Clostridial toxin in which 1) at least one amino acid is deletedfrom the reference Clostridial toxin on which the non-conservativeClostridial toxin variant is based; 2) at least one amino acid added tothe reference Clostridial toxin on which the non-conservativeClostridial toxin is based; or 3) at least one amino acid is substitutedby another amino acid or an amino acid analog that does not share anyproperty similar to that of the original amino acid from the referenceClostridial toxin sequence (Table 1). A non-conservative Clostridialtoxin variant can function in substantially the same manner as thereference Clostridial toxin on which the non-conservative Clostridialtoxin variant is based, and can be substituted for the referenceClostridial toxin in any aspect of the present invention. Anon-conservative Clostridial toxin variant can delete one or more aminoacids, two or more amino acids, three or more amino acids, four or moreamino acids, five or more amino acids, and ten or more amino acids fromthe reference Clostridial toxin on which the non-conservativeClostridial toxin variant is based. A non-conservative Clostridial toxinvariant can add one or more amino acids, two or more amino acids, threeor more amino acids, four or more amino acids, five or more amino acids,and ten or more amino acids to the reference Clostridial toxin on whichthe non-conservative Clostridial toxin variant is based. Anon-conservative Clostridial toxin variant may substitute one or moreamino acids, two or more amino acids, three or more amino acids, four ormore amino acids, five or more amino acids, ten or more amino acids, 20or more amino acids, 30 or more amino acids, 40 or more amino acids, 50or more amino acids, 100 or more amino acids, 200 or more amino acids,300 or more amino acids, 400 or more amino acids, or 500 or more aminoacids from the reference Clostridial toxin on which the non-conservativeClostridial toxin variant is based. A non-conservative Clostridial toxinvariant can also substitute at least 10 contiguous amino acids, at least15 contiguous amino acids, at least 20 contiguous amino acids, or atleast 25 contiguous amino acids from the reference Clostridial toxin onwhich the non-conservative Clostridial toxin variant is based, thatpossess at least 50% amino acid identity, 65% amino acid identity, 75%amino acid identity, 85% amino acid identity or 95% amino acid identityto the reference Clostridial toxin on which the non-conservativeClostridial toxin variant is based. Non-limiting examples of anon-conservative Clostridial toxin variant include, e.g.,non-conservative BoNT/A variants, non-conservative BoNT/B variants,non-conservative BoNT/C1 variants, non-conservative BoNT/D variants,non-conservative BoNT/E variants, non-conservative BoNT/F variants,non-conservative BoNT/G variants, and non-conservative TeNT variants.

As used herein, the term “Clostridial toxin chimeric variant” means amolecule comprising at least a portion of a Clostridial toxin and atleast a portion of at least one other protein to form a toxin with atleast one property different from the reference Clostridial toxins ofTable 1. Such Clostridial toxin chimeric molecules are described in,e.g., Clifford C. Shone et al., Recombinant Toxin Fragments, U.S. Pat.No. 6,461,617 (Oct. 8, 2002); Keith A. Foster et al., Clostridial ToxinDerivatives Able To Modify Peripheral Sensory Afferent Functions, U.S.Pat. No. 6,395,513 (May 28, 2002); Wei-Jin Lin et al., Neurotoxins withEnhanced Target Specificity, US 2002/0137886 (Sep. 26, 2002); Keith A.Foster et al., Inhibition of Secretion from Non-neural Cells, US2003/0180289 (Sep. 25, 2003); J. Oliver Dolly et al., ActivatableRecombinant Neurotoxins, WO 2001/014570 (Mar. 1, 2001); Clifford C.Shone et al., Recombinant Toxin Fragments, WO 2004/024909 (Mar. 25,2004); and Keith A. Foster et al., Re-targeted Toxin Conjugates, WO2005/023309 (Mar. 17, 2005).

It is well documented that toxin molecules can be re-targeted to a cellthat is not the toxins' natural target cell. When so re-targeted, thesetoxins are capable of binding to a desired target cell and, followingsubsequent translocation into the cytosol, are capable of exerting theireffect on the target cell. In this regard, the binding domain isselected so that it will bind to a desired target cell, and allowsubsequent passage of the modified Clostridial toxin into an endosomewithin the target cell. It is envisioned that any non-Clostridialbinding domain can be used, including, without limitation, ligands,hormones, growth factors, cytokines, antibodies, antagonists, agonistsand reverse-agonists, with the proviso that the non-Clostridial bindingdomain binds to a cell surface receptor system other than the one usedby the Clostridial binding domain of the modified Clostridial toxin.Non-limiting examples of a non-Clostridial binding domain include,growth factors, such as, e.g., Nerve growth factor (NGF), Leukemiainhibitory factor (LIF), Basic fibroblast growth factor (bFGF),Brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), Hydrahead activator peptide (HHAP), Transforming growth factor 1 (TGF-1),Transforming growth factor 2 (TGF-2), Transforming growth factor 3(TGF-3), Epidermal growth factor (EGF) and Ciliary neurotrophic factor(CNTF); cytokines, such as, e.g., Tumor necrosis factor (TNF-),Interleukin-1 (IL-1), Interleukin-1 (IL-1) and Interleukin-8 (IL-8);agonists, such as, e.g., Bradykinin, Dynorphin, β-endorphin, Etorphine,Endomorphin-1, Endomorphin-2Leu-enkephalin, Met-enkephalin, Galanin,Lofentanil, Nociceptin and an opioid; and antibodies, such as, e.g.,antibodies against the lactoseries carbohydrate epitopes found on thesurface of dorsal root ganglion neurons (e.g. monoclonal antibodies 1B2and LA4), antibodies against any of the receptors for the ligands givenabove and antibodies against the surface expressed antigen Thyl (e.g.monoclonal antibody MRC OX7). Methods of making and using a Clostridialtoxin chimeric variant can comprise a modified Clostridial toxindisclosed in the present specification where the binding domaincomprises a non-Clostridial toxin binding domain are described in, e.g.,Clifford C. Shone et al., supra, (2002); Keith A. Foster et al., supra,(2002); Wei-Jin Lin et al., supra, (2002); Keith A. Foster et al.,supra, (2003); J. Oliver Dolly et al., supra, (2001); Clifford C. Shoneet al., supra, (2004); and Keith A. Foster et al., supra, (2005).

Thus, in an embodiment, a Clostridial toxin chimeric variant cancomprise a modified Clostridial toxin disclosed in the presentspecification where the binding domain comprises a non-Clostridial toxinbinding domain. In aspects of this embodiment, a non-Clostridial toxinbinding domain can be, e.g., a ligand, a hormone, a growth factor, acytokine, an antibody, an opioid, an antagonist, an agonist or areverse-agonist. In other aspects of this embodiment, a non-Clostridialtoxin binding domain is a Nerve growth factor (NGF), a Leukemiainhibitory factor (LIF), a Basic fibroblast growth factor (bFGF), aBrain-derived neurotrophic factor (BDNF), a Neurotrophin-3 (NT-3), aHydra head activator peptide (HHAP), a Transforming growth factor 1(TGF-1), a Transforming growth factor 2 (TGF-2), a Transforming growthfactor 3 (TGF-3), an Epidermal growth factor (EGF) or a Ciliaryneurotrophic factor (CNTF). In still other aspects of this embodiment, anon-Clostridial toxin binding domain is a Tumor necrosis factor (TNF-),an Interleukin-1 (IL-1), an Interleukin-1 (IL-1) or an Interleukin-8(IL-8). In yet other aspects of this embodiment, a non-Clostridial toxinbinding domain is a Bradykinin, a Dynorphin, a β-endorphin, anEtorphine, an Endomorphin-1, an Endomorphin-2, a Leu-enkephalin, aMet-enkephalin, a Galanin, a Lofentanil or a Nociceptin. In still otheraspects of this embodiment, a non-Clostridial toxin binding domain is anantibody against the lactoseries carbohydrate epitopes found on thesurface of dorsal root ganglion neurons (e.g. monoclonal antibodies 1B2and LA4), an antibody against any of the receptors for the bindingdomains given above or an antibody against the surface expressed antigenThyl (e.g. monoclonal antibody MRC OX7).

It is also envisioned that any of a variety of Clostridial toxinfragments can be useful in aspects of the present invention with theproviso that these active fragments can execute the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. Thus, aspects of this embodiment can include Clostridialtoxin fragments having a length of, e.g., at least 300 amino acids, atleast 400 amino acids, at least 500 amino acids, at least 600 aminoacids, at least 700 amino acids, at least 800 amino acids, at least 900amino acids, at least 1000 amino acids, at least 1100 amino acids and atleast 1200 amino acids. Other aspects of this embodiment, can includeClostridial toxin fragments having a length of, e.g., at most 300 aminoacids, at most 400 amino acids, at most 500 amino acids, at most 600amino acids, at most 700 amino acids, at most 800 amino acids, at most900 amino acids, at most 1000 amino acids, at most 1100 amino acids andat most 1200 amino acids.

It is also envisioned that any of a variety of Clostridial toxinfragments comprising the light chain can be useful in aspects of thepresent invention with the proviso that these light chain fragments canspecifically target the core components of the neurotransmitter releaseapparatus and thus participate in executing the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate. The light chains of Clostridial toxins are approximately420-460 amino acids in length and comprise an enzymatic domain (Table1). Research has shown that the entire length of a Clostridial toxinlight chain is not necessary for the enzymatic activity of the enzymaticdomain. As a non-limiting example, the first eight amino acids of theBoNT/A light chain (residues 1-8 of SEQ ID NO: 1) are not required forenzymatic activity. As another non-limiting example, the first eightamino acids of the TeNT light chain (residues 1-8 of SEQ ID NO: 8) arenot required for enzymatic activity. Likewise, the carboxyl-terminus ofthe light chain is not necessary for activity. As a non-limitingexample, the last 32 amino acids of the BoNT/A light chain (residues417-448 of SEQ ID NO: 1) are not required for enzymatic activity. Asanother non-limiting example, the last 31 amino acids of the TeNT lightchain (residues 427-457 of SEQ ID NO: 8) are not required for enzymaticactivity. Thus, aspects of this embodiment can include Clostridial toxinlight chains comprising an enzymatic domain having a length of, e.g., atleast 350 amino acids, at least 375 amino acids, at least 400 aminoacids, at least 425 amino acids and at least 450 amino acids. Otheraspects of this embodiment can include Clostridial toxin light chainscomprising an enzymatic domain having a length of, e.g., at most 350amino acids, at most 375 amino acids, at most 400 amino acids, at most425 amino acids and at most 450 amino acids.

It is also envisioned that any of a variety of Clostridial toxin H_(N)regions comprising a translocation domain can be useful in aspects ofthe present invention with the proviso that these active fragments canfacilitate the release of the LC from intracellular vesicles into thecytoplasm of the target cell and thus participate in executing theoverall cellular mechanism whereby a Clostridial toxin proteolyticallycleaves a substrate. The H_(N) regions from the heavy chains ofClostridial toxins are approximately 410-430 amino acids in length andcomprise a translocation domain (Table 1). Research has shown that theentire length of a H_(N) region from a Clostridial toxin heavy chain isnot necessary for the translocating activity of the translocationdomain. Thus, aspects of this embodiment can include Clostridial toxinH_(N) regions comprising a translocation domain having a length of,e.g., at least 350 amino acids, at least 375 amino acids, at least 400amino acids and at least 425 amino acids. Other aspects of thisembodiment can include Clostridial toxin H_(N) regions comprisingtranslocation domain having a length of, e.g., at most 350 amino acids,at most 375 amino acids, at most 400 amino acids and at most 425 aminoacids.

It is also envisioned that any of a variety of Clostridial toxin H_(C)regions comprising a binding domain can be useful in aspects of thepresent invention with the proviso that these active fragments candetermine the binding activity and binding specificity of the toxin tothe receptor complex located at the surface of the target cell executethe overall cellular mechanism whereby a Clostridial toxinproteolytically cleaves a substrate. The H_(C) regions from the heavychains of Clostridial toxins are approximately 400-440 amino acids inlength and comprise a binding domain (Table 1). Research has shown thatthe entire length of a H_(C) region from a Clostridial toxin heavy chainis not necessary for the binding activity of the binding domain. Thus,aspects of this embodiment can include Clostridial toxin H_(C) regionscomprising a binding domain having a length of, e.g., at least 350 aminoacids, at least 375 amino acids, at least 400 amino acids and at least425 amino acids. Other aspects of this embodiment can includeClostridial toxin H_(C) regions comprising a binding domain having alength of, e.g., at most 350 amino acids, at most 375 amino acids, atmost 400 amino acids and at most 425 amino acids.

Thus, in an embodiment, a Clostridial toxin comprises a Clostridialtoxin enzymatic domain, a Clostridial toxin translocation domain and aClostridial toxin binding domain. In an aspect of this embodiment, aClostridial toxin comprises a naturally occurring Clostridial toxinvariant, such as, e.g., a Clostridial toxin isoform or a Clostridialtoxin subtype. In another aspect of this embodiment, a Clostridial toxincomprises a non-naturally occurring Clostridial toxin variant, such as,e.g., a conservative Clostridial toxin variant, a non-conservativeClostridial toxin variant or an active Clostridial toxin fragment, orany combination thereof. In another aspect of this embodiment, aClostridial toxin comprises a Clostridial toxin enzymatic domain or anactive fragment thereof, a Clostridial toxin translocation domain or anactive fragment thereof, a Clostridial toxin binding domain or an activefragment thereof, or any combination thereof. In other aspects of thisembodiment, a Clostridial toxin can comprise a BoNT/A, a BoNT/B, aBoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G or a TeNT.

In another embodiment, a Clostridial toxin comprises a BoNT/A. In anaspect of this embodiment, a BoNT/A comprises a BoNT/A enzymatic domain,a BoNT/A translocation domain and a BoNT/A binding domain. In anotheraspect of this embodiment, a BoNT/A comprises SEQ ID NO: 1. In anotheraspect of this embodiment, a BoNT/A comprises a naturally occurringBoNT/A variant, such as, e.g., a BoNT/A isoform or a BoNT/A subtype. Inanother aspect of this embodiment, a BoNT/A comprises a naturallyoccurring BoNT/A variant of SEQ ID NO: 1, such as, e.g., a BoNT/Aisoform of SEQ ID NO: 1 or a BoNT/A subtype of SEQ ID NO: 1. In stillanother aspect of this embodiment, a BoNT/A comprises a non-naturallyoccurring BoNT/A variant, such as, e.g., a conservative BoNT/A variant,a non-conservative BoNT/A variant or an active BoNT/A fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/A comprises a non-naturally occurring BoNT/A variant of SEQ ID NO:1, such as, e.g., a conservative BoNT/A variant of SEQ ID NO: 1, anon-conservative BoNT/A variant of SEQ ID NO: 1 or an active BoNT/Afragment of SEQ ID NO: 1, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/A comprises a BoNT/A enzymatic domainor an active fragment thereof, a BoNT/A translocation domain or anactive fragment thereof, a BoNT/A binding domain or an active fragmentthereof, or any combination thereof. In yet another aspect of thisembodiment, a BoNT/A comprising a BoNT/A enzymatic domain of amino acids1-448 from SEQ ID NO: 1 or an active fragment thereof, a BoNT/Atranslocation domain of amino acids 449-871 from SEQ ID NO: 1 or anactive fragment thereof, a BoNT/A binding domain of amino acids 872-1296from SEQ ID NO: 1 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a BoNT/A comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 1, atleast 75% amino acid identity with the SEQ ID NO: 1, at least 80% aminoacid identity with SEQ ID NO: 1, at least 85% amino acid identity withSEQ ID NO: 1, at least 90% amino acid identity with SEQ ID NO: 1 or atleast 95% amino acid identity with SEQ ID NO: 1. In yet other aspects ofthis embodiment, a BoNT/A comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 1, at most 75% amino acididentity with the SEQ ID NO: 1, at most 80% amino acid identity with SEQID NO: 1, at most 85% amino acid identity with SEQ ID NO: 1, at most 90%amino acid identity with SEQ ID NO: 1 or at most 95% amino acid identitywith SEQ ID NO: 1.

In other aspects of this embodiment, a BoNT/A comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 1. In yet other aspects of this embodiment, a BoNT/A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 1. In still other aspects of this embodiment, a BoNT/A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 1.

In other aspects of this embodiment, a BoNT/A comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 1. In yet other aspects of this embodiment, a BoNT/A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ IDNO: 1. In still other aspects of this embodiment, a BoNT/A comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 1. In other aspects of thisembodiment, a BoNT/A comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:1.

In another embodiment, a Clostridial toxin comprises a BoNT/B. In anaspect of this embodiment, a BoNT/B comprises a BoNT/B enzymatic domain,a BoNT/B translocation domain and a BoNT/B binding domain. In anotheraspect of this embodiment, a BoNT/B comprises SEQ ID NO: 2. In anotheraspect of this embodiment, a BoNT/B comprises a naturally occurringBoNT/B variant, such as, e.g., a BoNT/B isoform or a BoNT/B subtype. Inanother aspect of this embodiment, a BoNT/B comprises a naturallyoccurring BoNT/B variant of SEQ ID NO: 2, such as, e.g., a BoNT/Bisoform of SEQ ID NO: 2 or a BoNT/B subtype of SEQ ID NO: 2. In stillanother aspect of this embodiment, a BoNT/B comprises a non-naturallyoccurring BoNT/B variant, such as, e.g., a conservative BoNT/B variant,a non-conservative BoNT/B variant or an active BoNT/B fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/B comprises a non-naturally occurring BoNT/B variant of SEQ ID NO:2, such as, e.g., a conservative BoNT/B variant of SEQ ID NO: 2, anon-conservative BoNT/B variant of SEQ ID NO: 2 or an active BoNT/Bfragment of SEQ ID NO: 2, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/B comprising a BoNT/B enzymatic domainor an active fragment thereof, a BoNT/B translocation domain or activefragment thereof, a BoNT/B binding domain or active fragment thereof,and any combination thereof. In yet another aspect of this embodiment, aBoNT/B comprising a BoNT/B enzymatic domain of amino acids 1-441 fromSEQ ID NO: 2 or active fragment thereof, a BoNT/B translocation domainof amino acids 442-858 from SEQ ID NO: 2 or active fragment thereof, aBoNT/B binding domain of amino acids 859-1291 from SEQ ID NO: 2 oractive fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/B comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 2, atleast 75% amino acid identity with the SEQ ID NO: 2, at least 80% aminoacid identity with SEQ ID NO: 2, at least 85% amino acid identity withSEQ ID NO: 2, at least 90% amino acid identity with SEQ ID NO: 2 or atleast 95% amino acid identity with SEQ ID NO: 2. In yet other aspects ofthis embodiment, a BoNT/B comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 2, at most 75% amino acididentity with the SEQ ID NO: 2, at most 80% amino acid identity with SEQID NO: 2, at most 85% amino acid identity with SEQ ID NO: 2, at most 90%amino acid identity with SEQ ID NO: 2 or at most 95% amino acid identitywith SEQ ID NO: 2.

In other aspects of this embodiment, a BoNT/B comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 2. In yet other aspects of this embodiment, a BoNT/B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 2. In still other aspects of this embodiment, a BoNT/B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 2.

In other aspects of this embodiment, a BoNT/B comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 2. In yet other aspects of this embodiment, a BoNT/B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:2. In still other aspects of this embodiment, a BoNT/B comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 2. In other aspects of thisembodiment, a BoNT/B comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:2.

In another embodiment, a Clostridial toxin comprises a BoNT/C1. In anaspect of this embodiment, a BoNT/C1 comprises a BoNT/C1 enzymaticdomain, a BoNT/C1 translocation domain and a BoNT/C1 binding domain. Inanother aspect of this embodiment, a BoNT/C1 comprises SEQ ID NO: 3. Inanother aspect of this embodiment, a BoNT/C1 comprises a naturallyoccurring BoNT/C1 variant, such as, e.g., a BoNT/C1 isoform or a BoNT/C1subtype. In another aspect of this embodiment, a BoNT/C1 comprises anaturally occurring BoNT/C1 variant of SEQ ID NO: 3, such as, e.g., aBoNT/C1 isoform of SEQ ID NO: 3 or a BoNT/C1 subtype of SEQ ID NO: 3. Instill another aspect of this embodiment, a BoNT/C1 comprises anon-naturally occurring BoNT/C1 variant, such as, e.g., a conservativeBoNT/C1 variant, a non-conservative BoNT/C1 variant or an active BoNT/C1fragment, or any combination thereof. In still another aspect of thisembodiment, a BoNT/C1 comprises a non-naturally occurring BoNT/C1variant of SEQ ID NO: 3, such as, e.g., a conservative BoNT/C1 variantof SEQ ID NO: 3, a non-conservative BoNT/C1 variant of SEQ ID NO: 3 oran active BoNT/C1 fragment of SEQ ID NO: 3, or any combination thereof.In yet another aspect of this embodiment, a BoNT/C1 comprises a BoNT/C1enzymatic domain or active fragment thereof, a BoNT/C1 translocationdomain or active fragment thereof, a BoNT/C1 binding domain or activefragment thereof, and any combination thereof. In yet another aspect ofthis embodiment, a BoNT/C1 comprises a BoNT/C1 enzymatic domain of aminoacid 1-449 from SEQ ID NO: 3 or active fragment thereof, a BoNT/C1translocation domain of amino acids 450-866 from SEQ ID NO: 3 or activefragment thereof, a BoNT/C1 binding domain of amino acids 867-1291 fromSEQ ID NO: 3 or active fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 3, atleast 75% amino acid identity with the SEQ ID NO: 3, at least 80% aminoacid identity with SEQ ID NO: 3, at least 85% amino acid identity withSEQ ID NO: 3, at least 90% amino acid identity with SEQ ID NO: 3 or atleast 95% amino acid identity with SEQ ID NO: 3. In yet other aspects ofthis embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 3, at most 75% amino acididentity with the SEQ ID NO: 3, at most 80% amino acid identity with SEQID NO: 3, at most 85% amino acid identity with SEQ ID NO: 3, at most 90%amino acid identity with SEQ ID NO: 3 or at most 95% amino acid identitywith SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, 200 or 500 non-contiguous amino acid substitutions relative toSEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1comprises a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid deletionsrelative to SEQ ID NO: 3. In still other aspects of this embodiment, aBoNT/C1 comprises a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 3. In otheraspects of this embodiment, a BoNT/C1 comprises a polypeptide having,e.g., at least one, two, three, four, five, six, seven, eight, nine, 10,20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid additionsrelative to SEQ ID NO: 3.

In other aspects of this embodiment, a BoNT/C1 comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, 200 or 500 contiguous amino acid substitutions relative to SEQID NO: 3. In yet other aspects of this embodiment, a BoNT/C1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, 200 or 500 contiguous amino acid deletions relative to SEQ IDNO: 3. In still other aspects of this embodiment, a BoNT/C1 comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 3. In other aspects of thisembodiment, a BoNT/C1 comprises a polypeptide having, e.g., at leastone, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,50, 100, 200 or 500 contiguous amino acid additions relative to SEQ IDNO: 3.

In another embodiment, a Clostridial toxin comprises a BoNT/D. In anaspect of this embodiment, a BoNT/D comprises a BoNT/D enzymatic domain,a BoNT/D translocation domain and a BoNT/D binding domain. In anotheraspect of this embodiment, a BoNT/D comprises SEQ ID NO: 4. In anotheraspect of this embodiment, a BoNT/D comprises a naturally occurringBoNT/D variant, such as, e.g., a BoNT/D isoform or a BoNT/D subtype. Inanother aspect of this embodiment, a BoNT/D comprises a naturallyoccurring BoNT/D variant of SEQ ID NO: 4, such as, e.g., a BoNT/Disoform of SEQ ID NO: 4 or a BoNT/D subtype of SEQ ID NO: 4. In stillanother aspect of this embodiment, a BoNT/D comprises a non-naturallyoccurring BoNT/D variant, such as, e.g., a conservative BoNT/D variant,a non-conservative BoNT/D variant or an active BoNT/D fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/D comprises a non-naturally occurring BoNT/D variant of SEQ ID NO:4, such as, e.g., a conservative BoNT/D variant of SEQ ID NO: 4, anon-conservative BoNT/D variant of SEQ ID NO: 4 or an active BoNT/Dfragment of SEQ ID NO: 4, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/D comprises a BoNT/D enzymatic domainor an active fragment thereof, a BoNT/D translocation domain or anactive fragment thereof, a BoNT/D binding domain or an active fragmentthereof, or any combination thereof. In yet another aspect of thisembodiment, a BoNT/D comprising a BoNT/D enzymatic domain of amino acids1-445 from SEQ ID NO: 4 or an active fragment thereof, a BoNT/Dtranslocation domain of amino acids 446-862 from SEQ ID NO: 4 or anactive fragment thereof, a BoNT/D binding domain of amino acids 863-1276from SEQ ID NO: 4 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a BoNT/D comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 4, atleast 75% amino acid identity with the SEQ ID NO: 4, at least 80% aminoacid identity with SEQ ID NO: 4, at least 85% amino acid identity withSEQ ID NO: 4, at least 90% amino acid identity with SEQ ID NO: 4 or atleast 95% amino acid identity with SEQ ID NO: 4. In yet other aspects ofthis embodiment, a BoNT/D comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 4, at most 75% amino acididentity with the SEQ ID NO: 4, at most 80% amino acid identity with SEQID NO: 4, at most 85% amino acid identity with SEQ ID NO: 4, at most 90%amino acid identity with SEQ ID NO: 4 or at most 95% amino acid identitywith SEQ ID NO: 4.

In other aspects of this embodiment, a BoNT/D comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 4. In yet other aspects of this embodiment, a BoNT/D comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 4. In still other aspects of this embodiment, a BoNT/D comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 4.

In other aspects of this embodiment, a BoNT/D comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 4. In yet other aspects of this embodiment, a BoNT/D comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:4. In still other aspects of this embodiment, a BoNT/D comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 4. In other aspects of thisembodiment, a BoNT/D comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:4.

In another embodiment, a Clostridial toxin comprises a BoNT/E. In anaspect of this embodiment, a BoNT/E comprises a BoNT/E enzymatic domain,a BoNT/E translocation domain and a BoNT/E binding domain. In anotheraspect of this embodiment, a BoNT/E comprises SEQ ID NO: 5. In anotheraspect of this embodiment, a BoNT/E comprises a naturally occurringBoNT/E variant, such as, e.g., a BoNT/E isoform or a BoNT/E subtype. Inanother aspect of this embodiment, a BoNT/E comprises a naturallyoccurring BoNT/E variant of SEQ ID NO: 5, such as, e.g., a BoNT/Eisoform of SEQ ID NO: 5 or a BoNT/E subtype of SEQ ID NO: 5. In stillanother aspect of this embodiment, a BoNT/E comprises a non-naturallyoccurring BoNT/E variant, such as, e.g., a conservative BoNT/E variant,a non-conservative BoNT/E variant or an active BoNT/E fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/E comprises a non-naturally occurring BoNT/E variant of SEQ ID NO:5, such as, e.g., a conservative BoNT/E variant of SEQ ID NO: 5, anon-conservative BoNT/E variant of SEQ ID NO: 5 or an active BoNT/Efragment of SEQ ID NO: 5, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/E comprising a BoNT/E enzymatic domainor an active fragment thereof, a BoNT/E translocation domain or activefragment thereof, a BoNT/E binding domain or active fragment thereof,and any combination thereof. In yet another aspect of this embodiment, aBoNT/E comprising a BoNT/E enzymatic domain of amino acids 1-422 fromSEQ ID NO: 5 or active fragment thereof, a BoNT/E translocation domainof amino acids 423-845 from SEQ ID NO: 5 or active fragment thereof, aBoNT/E binding domain of amino acids 846-1252 from SEQ ID NO: 5 oractive fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/E comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 5, atleast 75% amino acid identity with the SEQ ID NO: 5, at least 80% aminoacid identity with SEQ ID NO: 5, at least 85% amino acid identity withSEQ ID NO: 5, at least 90% amino acid identity with SEQ ID NO: 5 or atleast 95% amino acid identity with SEQ ID NO: 5. In yet other aspects ofthis embodiment, a BoNT/E comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 5, at most 75% amino acididentity with the SEQ ID NO: 5, at most 80% amino acid identity with SEQID NO: 5, at most 85% amino acid identity with SEQ ID NO: 5, at most 90%amino acid identity with SEQ ID NO: 5 or at most 95% amino acid identitywith SEQ ID NO: 5.

In other aspects of this embodiment, a BoNT/E comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 5. In yet other aspects of this embodiment, a BoNT/E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 5. In still other aspects of this embodiment, a BoNT/E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 5.

In other aspects of this embodiment, a BoNT/E comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 5. In yet other aspects of this embodiment, a BoNT/E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:5. In still other aspects of this embodiment, a BoNT/E comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 5. In other aspects of thisembodiment, a BoNT/E comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:5.

In another embodiment, a Clostridial toxin comprises a BoNT/F. In anaspect of this embodiment, a BoNT/F comprises a BoNT/F enzymatic domain,a BoNT/F translocation domain and a BoNT/F binding domain. In anotheraspect of this embodiment, a BoNT/F comprises SEQ ID NO: 6. In anotheraspect of this embodiment, a BoNT/F comprises a naturally occurringBoNT/F variant, such as, e.g., a BoNT/F isoform or a BoNT/F subtype. Inanother aspect of this embodiment, a BoNT/F comprises a naturallyoccurring BoNT/F variant of SEQ ID NO: 6, such as, e.g., a BoNT/Fisoform of SEQ ID NO: 6 or a BoNT/F subtype of SEQ ID NO: 6. In stillanother aspect of this embodiment, a BoNT/F comprises a non-naturallyoccurring BoNT/F variant, such as, e.g., a conservative BoNT/F variant,a non-conservative BoNT/F variant or an active BoNT/F fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/F comprises a non-naturally occurring BoNT/F variant of SEQ ID NO:6, such as, e.g., a conservative BoNT/F variant of SEQ ID NO: 6, anon-conservative BoNT/F variant of SEQ ID NO: 6 or an active BoNT/Ffragment of SEQ ID NO: 6, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/F comprises a BoNT/F enzymatic domainor active fragment thereof, a BoNT/F translocation domain or activefragment thereof, a BoNT/F binding domain or active fragment thereof,and any combination thereof. In yet another aspect of this embodiment, aBoNT/F comprises a BoNT/F enzymatic domain of amino acid 1-439 from SEQID NO: 6 or active fragment thereof, a BoNT/F translocation domain ofamino acids 440-864 from SEQ ID NO: 6 or active fragment thereof, aBoNT/F binding domain of amino acids 865-1274 from SEQ ID NO: 6 oractive fragment thereof, and any combination thereof.

In other aspects of this embodiment, a BoNT/F comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 6, atleast 75% amino acid identity with the SEQ ID NO: 6, at least 80% aminoacid identity with SEQ ID NO: 6, at least 85% amino acid identity withSEQ ID NO: 6, at least 90% amino acid identity with SEQ ID NO: 6 or atleast 95% amino acid identity with SEQ ID NO: 6. In yet other aspects ofthis embodiment, a BoNT/F comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 6, at most 75% amino acididentity with the SEQ ID NO: 6, at most 80% amino acid identity with SEQID NO: 6, at most 85% amino acid identity with SEQ ID NO: 6, at most 90%amino acid identity with SEQ ID NO: 6 or at most 95% amino acid identitywith SEQ ID NO: 6.

In other aspects of this embodiment, a BoNT/F comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 6. In yet other aspects of this embodiment, a BoNT/F comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 6. In still other aspects of this embodiment, a BoNT/F comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 6.

In other aspects of this embodiment, a BoNT/F comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 6. In yet other aspects of this embodiment, a BoNT/F comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:6. In still other aspects of this embodiment, a BoNT/F comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 6. In other aspects of thisembodiment, a BoNT/F comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:6.

In another embodiment, a Clostridial toxin comprises a BoNT/G. In anaspect of this embodiment, a BoNT/G comprises a BoNT/G enzymatic domain,a BoNT/G translocation domain and a BoNT/G binding domain. In anotheraspect of this embodiment, a BoNT/G comprises SEQ ID NO: 7. In anotheraspect of this embodiment, a BoNT/G comprises a naturally occurringBoNT/G variant, such as, e.g., a BoNT/G isoform or a BoNT/G subtype. Inanother aspect of this embodiment, a BoNT/G comprises a naturallyoccurring BoNT/G variant of SEQ ID NO: 7, such as, e.g., a BoNT/Gisoform of SEQ ID NO: 7 or a BoNT/G subtype of SEQ ID NO: 7. In stillanother aspect of this embodiment, a BoNT/G comprises a non-naturallyoccurring BoNT/G variant, such as, e.g., a conservative BoNT/G variant,a non-conservative BoNT/G variant or an active BoNT/G fragment, or anycombination thereof. In still another aspect of this embodiment, aBoNT/D comprises a non-naturally occurring BoNT/G variant of SEQ ID NO:7, such as, e.g., a conservative BoNT/G variant of SEQ ID NO: 7, anon-conservative BoNT/G variant of SEQ ID NO: 7 or an active BoNT/Gfragment of SEQ ID NO: 7, or any combination thereof. In yet anotheraspect of this embodiment, a BoNT/G comprises a BoNT/G enzymatic domainor an active fragment thereof, a BoNT/G translocation domain or anactive fragment thereof, a BoNT/G binding domain or an active fragmentthereof, or any combination thereof. In yet another aspect of thisembodiment, a BoNT/G comprising a BoNT/G enzymatic domain of amino acids1-446 from SEQ ID NO: 7 or an active fragment thereof, a BoNT/Gtranslocation domain of amino acids 447-863 from SEQ ID NO: 7 or anactive fragment thereof, a BoNT/G binding domain of amino acids 864-1297from SEQ ID NO: 7 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a BoNT/G comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 7, atleast 75% amino acid identity with the SEQ ID NO: 7, at least 80% aminoacid identity with SEQ ID NO: 7, at least 85% amino acid identity withSEQ ID NO: 7, at least 90% amino acid identity with SEQ ID NO: 7 or atleast 95% amino acid identity with SEQ ID NO: 7. In yet other aspects ofthis embodiment, a BoNT/G comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 7, at most 75% amino acididentity with the SEQ ID NO: 7, at most 80% amino acid identity with SEQID NO: 7, at most 85% amino acid identity with SEQ ID NO: 7, at most 90%amino acid identity with SEQ ID NO: 7 or at most 95% amino acid identitywith SEQ ID NO: 7.

In other aspects of this embodiment, a BoNT/G comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 7. In yet other aspects of this embodiment, a BoNT/G comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 7. In still other aspects of this embodiment, a BoNT/G comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 7.

In other aspects of this embodiment, a BoNT/G comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 7. In yet other aspects of this embodiment, a BoNT/G comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:7. In still other aspects of this embodiment, a BoNT/G comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 7. In other aspects of thisembodiment, a BoNT/G comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:7.

In another embodiment, a Clostridial toxin comprises a TeNT. In anaspect of this embodiment, a TeNT comprises a TeNT enzymatic domain, aTeNT translocation domain and a TeNT binding domain. In an aspect ofthis embodiment, a TeNT comprises SEQ ID NO: 8. In another aspect ofthis embodiment, a TeNT comprises a naturally occurring TeNT variant,such as, e.g., a TeNT isoform or a TeNT subtype. In another aspect ofthis embodiment, a TeNT comprises a naturally occurring TeNT variant ofSEQ ID NO: 8, such as, e.g., a TeNT isoform of SEQ ID NO: 8 or a TeNTsubtype of SEQ ID NO: 8. In still another aspect of this embodiment, aTeNT comprises a non-naturally occurring TeNT variant, such as, e.g., aconservative TeNT variant, a non-conservative TeNT variant or an activeTeNT fragment, or any combination thereof. In still another aspect ofthis embodiment, a TeNT comprises a non-naturally occurring TeNT variantof SEQ ID NO: 8, such as, e.g., a conservative TeNT variant of SEQ IDNO: 8, a non-conservative TeNT variant of SEQ ID NO: 8 or an active TeNTfragment of SEQ ID NO: 8, or any combination thereof. In yet anotheraspect of this embodiment, a TeNT comprising a TeNT enzymatic domain oran active fragment thereof, a TeNT translocation domain or activefragment thereof, a TeNT binding domain or active fragment thereof, andany combination thereof. In yet another aspect of this embodiment, aTeNT comprising a TeNT enzymatic domain of amino acids 1-457 from SEQ IDNO: 8 or active fragment thereof, a TeNT translocation domain of aminoacids 458-879 from SEQ ID NO: 8 or active fragment thereof, a TeNTbinding domain of amino acids 880-1315 from SEQ ID NO: 8 or activefragment thereof, and any combination thereof.

In other aspects of this embodiment, a TeNT comprises a polypeptidehaving, e.g., at least 70% amino acid identity with SEQ ID NO: 8, atleast 75% amino acid identity with the SEQ ID NO: 8, at least 80% aminoacid identity with SEQ ID NO: 8, at least 85% amino acid identity withSEQ ID NO: 8, at least 90% amino acid identity with SEQ ID NO: 8 or atleast 95% amino acid identity with SEQ ID NO: 8. In yet other aspects ofthis embodiment, a TeNT comprises a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 8, at most 75% amino acididentity with the SEQ ID NO: 8, at most 80% amino acid identity with SEQID NO: 8, at most 85% amino acid identity with SEQ ID NO: 8, at most 90%amino acid identity with SEQ ID NO: 8 or at most 95% amino acid identitywith SEQ ID NO: 8.

In other aspects of this embodiment, a TeNT comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acidsubstitutions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid substitutions relative to SEQID NO: 8. In yet other aspects of this embodiment, a TeNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid deletions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid deletions relative to SEQ IDNO: 8. In still other aspects of this embodiment, a TeNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguousamino acid additions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 non-contiguous amino acid additions relative to SEQ IDNO: 8.

In other aspects of this embodiment, a TeNT comprises a polypeptidehaving, e.g., at most one, two, three, four, five, six, seven, eight,nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acidsubstitutions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid substitutions relative to SEQ IDNO: 8. In yet other aspects of this embodiment, a TeNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid deletions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid deletions relative to SEQ ID NO:8. In still other aspects of this embodiment, a TeNT comprises apolypeptide having, e.g., at most one, two, three, four, five, six,seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous aminoacid additions relative to SEQ ID NO: 8. In other aspects of thisembodiment, a TeNT comprises a polypeptide having, e.g., at least one,two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,100, 200 or 500 contiguous amino acid additions relative to SEQ ID NO:8.

Aspects of the present invention provide, in part, a PAR ligand domain.As used herein, the term “PAR ligand domain” is synonymous with“tethered ligand” and “activating peptide” and means any polypeptidethat can selectively bind to the PAR ligand binding domain and initiatethe overall internalization mechanism whereby an activated PAR isinternalized into a cell. As used herein, the term “selectively” meanshaving a unique effect or influence or reacting in only one way or withonly one thing. As used herein, the term “selectively bind” means that aPAR ligand domain is able to bind its target PAR ligand binding domainunder physiological conditions, or in vitro conditions substantiallyapproximating physiological conditions, to a statistically significantlygreater degree (i.e., has a smaller Kd or dissociation constant) than toother, non-target ligand binding domains. “Kd” is the molarconcentration of the PAR ligand domain at which half the PAR ligandbinding domains are bound by the PAR ligand domain. Thus, there is adiscriminatory binding of the PAR ligand domain to the indicated targetbinding site.

Most G protein-coupled receptors (GPCRs) are reversibly activated uponligand binding. However, activation of protease-activated Gprotein-coupled receptors (PARs) occurs through an irreversibleproteolytic event that results in the generation of a tethered ligandthat cannot diffuse away. In essence, PARs are receptors that carrytheir own ligands, which remain unbound until unmasked by site-specificreceptor cleavage. The coagulant protease Thrombin is the physiologicalactivator of PAR1, PAR3 and PAR4; however, other proteases can cleavethese receptors and may contribute to their function in vivo (Table 2).PAR2 is activated by multiple Trypsin-like serine proteases includingTrypsin, Tryptase and coagulation proteases upstream of Thrombin,Factors VIIa and Xa, but not by Thrombin (Table 2).

Currently four subtypes of human PARs are described and designated PAR1(SEQ ID NO: 9), PAR2 (SEQ ID NO: 10), PAR3 (SEQ ID NO: 11) and PAR4 (SEQID NO: 12). In addition, PAR1, PAR2, PAR3 and PAR4 orthologs whichexhibit at least 70% amino acid identity and at least 80% amino acidsimilarity have been identified in other mammals, such as, e.g., thechimpanzee Pan troglodytes, the hamadryas baboon Papio hamadryas, thedog Canis familiaris, the mouse Mus musculus, the rat Rattus norvegicusand the chicken Gallus gallus. The protease cleavage site, which uponcleavage unmasks the tethered ligand, is known for all four receptors(Table 2). In human PARs, cleavage of PAR1 at R41-S42 exposes a newamino terminus ending in the hexapeptide SFLLRN, cleavage of PAR2 atR34-S35 exposes a new amino terminus ending in the hexapeptide SLIGKV,cleavage of PAR3 at K38-T39 exposes a new amino terminus ending in thehexapeptide TFRGAP, where as, cleavage of PAR4 at R47-G48 exposes a newamino terminus ending in the hexapeptide GYPGQV. A hirudin-like sitedistal to the protease cleavage site has been described in PAR1 andPAR3. This charged domain appears to help mediate the binding ofThrombin to PAR1, thereby facilitating cleavage of the protease cleavagesite.

TABLE 2 Summary of the Human PAR Family PAR1 PAR2 PAR3 PAR4 EndogenousARC Acrosien Thrombin Cathepsin G Activating Factor Xa Factor Xa FactorXa proteases Thrombin Factor VIIa Factor VIIa Trypsins MT-SP1 PlasminProteinase-3 Thrombin Trypsins Trypsins Tryptases Exogenous Granzyme ADer P1 Gingipains-R Activating Gingipains-R Der P3 proteases Der P9Gingipains-R Inactivating Cathepsin G Cathepsin G Cathepsin G proteasesElastase Elastase Plasmin Proteinase-3 Trypsins Cleavage siteLDPR⁴¹*S⁴²FLLRN SKGR³⁴*S³⁵LIGKV LPIK³⁸*T³⁹FRGAP PAPR⁴⁷*G⁴⁸YPGQVLocalization platelets epithelium platelets platelets endotheliumendothelium endothelium endothelium epithelium fibroblasts myocytesmyocytes fibroblasts myocytes astrocytes astrocytes myocytes neuronsneurons astrocytes astrocytes An asterisks (*) indicates the peptidebond that is cleaved by an activating PAR protease.

Synthetic peptides representing the newly formed amino terminus tetheredligand of PARs can act as agonists for the receptor without the need forproteolysis and can initiate many of the same signaling responseselicited by proteolytically activated PARs (Table 3), see e.g., Shaun R.Coughlin and Mark Kahn, Modulation of Platelet Activation, PCT PatentPublication WO 01/07072 (Feb. 1, 2001); Shaun R. Coughlin and Tatjana R.Faruqi, Peptides Modulating Protease Activated Receptors and Methods ofUsing Same, PCT Patent Publication WO 01/94411 (Dec. 13, 2001); Scott R.MacFarlane et al., Protease-Activated Receptors, 53 (2) Pharmacol. Rev.245-282 (2001); and Robert M. Scarborough, Protease-Activated Receptor-2Antagonists and Agonists, 1 (1) Curr. Med. Chem. Cardiovasc. Hematol.Agents 73-82 (2003). Referred to as activating peptides (AP), thesepeptides evoke the ligand binding, the signal transduction and thesignal termination steps described above. The first described AP was the14-residue peptide SFLLRNPNDKYEPF comprising amino acids 42-55 of SEQ IDNO: 13 that behaves as an agonist for PAR1. Subsequent work has shownthat not only the hexapeptide SFLLRN, but a wide range of variants werealso effective, if not fully functional to elicit a cellular response(Table 3). Analysis of PAR APs using alanine scanning and site-directedmutagenesis has identified residues critical for function. For example,the residues F2, L4 and R5 are functionally important for the PAR1 APhexapeptide SFLLRN, but substitutions of residues at other positions canbe tolerated. Similar testing of the PAR2 AP hexapeptide SLIGKVindicates that L2 and R5 are essential for PAR2 AP activity whereassubstitution of G4 or L6 has only a slight effect on PAR2 activation.Replacing S1 or I3 with alanine also reduces activity. While many PAR4variants are functional (Table 3), the specificity of PAR4 AP requiresY2, since replacement with F generates an agonist of both PAR1 and PAR4.

TABLE 3 PAR Binding Domains Amino Acid Sequence SEQ ID NO: PAR1Reference SFLLRN 13 Variants SFFLRN 14 SFFLKN 133 TFLLRN 15 GFPGKF 16GYPAKF 17 GYPLKF 18 GYPIKF 19 G(F)PGKF 20 GYP(Cha)KF 21 S(F)(Cha)(Cha)RK22 S(F)(Cha)(Cha)(homoR)K 23 PAR2 Reference SLIGKV 24 Variants SLIGRL 25PAR3 Reference TFRGAP 26 Variants SFNGGP 27 SFNGNE 134 PAR4 ReferenceGYPGQV 28 Variants AYPGKF 29 TYPGKF 30 GYPGKY 31 GYPGKW 32 GYPGKK 33GYPGKF 34 GYPGRF 35 GYPGFK 36 GYPAKF 37 GFPGKF 38 GFPGKP 39 SYPGKF 40SYPAKF 41 SYPGRF 42 SYAGKF 43 SFPGQP 135 SFPGQA 160 GYPG(Orn)F 44G(F)PGKF 45 GYPG(homoR)F 46 SYPG(homoR)F 47 (Cha), cyclohexylalanine;(homoR), homoarginine; (Orn), ornithine; (F), parafluoro-phenylalanine;other letters represent the single letter amino acid code.

It is envisioned that any and all PAR ligand domains capable of bindingan inactivated PAR and eliciting the internalization of the modifiedClostridial toxin-PAR complex into a cell can be useful in aspects ofthe present invention. It is envisioned that a PAR ligand domain of anyand all lengths can be useful in aspects of the present invention withthe proviso that the PAR ligand domain is capable of binding aninactivated PAR and eliciting the internalization of the modifiedClostridial toxin-PAR complex into a cell. Thus, in aspects of thisembodiment, a PAR ligand domain can be, e.g., at least 6 amino acids inlength, at least 7 amino acids in length, at least 8 amino acids inlength, at least 9 amino acids in length, at least 10 amino acids inlength, at least 15 amino acids in length, at least 20 amino acids inlength, at least 25 amino acids in length, at least 30 amino acids inlength, at least 40 amino acids in length, at least 50 amino acids inlength or at least 60 amino acids in length. In other aspects of thisembodiment, a PAR ligand domain can be, e.g., at most 6 amino acids inlength, at most 7 amino acids in length, at most 8 amino acids inlength, at most 9 amino acids in length, at most 10 amino acids inlength, at most 15 amino acids in length, at most 20 amino acids inlength, at most 25 amino acids in length, at most 30 amino acids inlength, at most 40 amino acids in length, at most 50 amino acids inlength or at most 60 amino acids in length. As a non-limiting example, aPAR 1 ligand domain can comprise amino acids 1-64 of SEQ ID NO: 9, aminoacids 1-55 of SEQ ID NO: 9, amino acids 1-47 of SEQ ID NO: 9, aminoacids 29-64 of SEQ ID NO: 9, amino acids 42-55 of SEQ ID NO: 9 or aminoacids 42-47 of SEQ ID NO: 9. As another non-limiting example, a PAR 2ligand domain can comprise amino acids 1-59 of SEQ ID NO: 10, compriseamino acids 1-48 of SEQ ID NO: 10, comprise amino acids 1-40 of SEQ IDNO: 10, amino acids 24-59 of SEQ ID NO: 10, amino acids 35-48 of SEQ IDNO: 10 or amino acids 35-40 of SEQ ID NO: 10. As still anothernon-limiting example, a PAR 3 ligand domain can comprise amino acids1-60 of SEQ ID NO: 11, comprise amino acids 1-52 of SEQ ID NO: 11,comprise amino acids 1-44 of SEQ ID NO: 11, amino acids 26-60 of SEQ IDNO: 11, amino acids 39-52 of SEQ ID NO: 11 or amino acids 39-44 of SEQID NO: 11. As yet another non-limiting example, a PAR 4 ligand domaincan comprise amino acids 1-70 of SEQ ID NO: 12, comprise amino acids1-61 of SEQ ID NO: 12, comprise amino acids 1-53 of SEQ ID NO: 12, aminoacids 35-70 of SEQ ID NO: 12, amino acids 48-61 of SEQ ID NO: 12 oramino acids 48-53 of SEQ ID NO: 12.

A PAR ligand domain useful in aspects of the invention includes, withoutlimitation, naturally occurring PAR ligand domains, such as, e.g., aPAR1 tethered ligand, a PAR2 tethered ligand, a PAR3 tethered ligand ora PAR4 tethered ligand; naturally occurring PAR ligand domain variants;and non-naturally-occurring PAR ligand domain variants, such as, e.g.,conservative PAR ligand domain variants, non-conservative PAR liganddomain variants and PAR ligand domain peptidomimetics. As used herein,the term “PAR ligand domain variant,” whether naturally-occurring ornon-naturally-occurring, means a PAR ligand domain that has at least oneamino acid change from the corresponding region of the disclosedreference sequences and can be described in percent identity to thecorresponding region of that reference sequence (Table 3). Any of avariety of sequence alignment methods can be used to determine percentidentity, including, without limitation, global methods, local methodsand hybrid methods, such as, e.g., segment approach methods. Protocolsto determine percent identity are routine procedures within the scope ofone skilled in the art and from the teaching herein.

As used herein, the term “naturally occurring PAR ligand domain variant”means any PAR ligand domain produced without the aid of any humanmanipulation, including, without limitation, PAR ligand domain isoformsproduced from alternatively-spliced transcripts, PAR ligand domainisoforms produced by spontaneous mutation and PAR ligand domainsubtypes.

As used herein, the term “non-naturally occurring PAR ligand domainvariant” means any PAR ligand domain produced with the aid of humanmanipulation, including, without limitation, PAR ligand domain variantsproduced by genetic engineering using random mutagenesis or rationaldesign and PAR ligand domain variants produced by chemical synthesis.Non-limiting examples of non-naturally occurring PAR ligand domainvariant include, e.g., conservative PAR ligand domain variants,non-conservative PAR ligand domain variants and PAR ligand domainpeptidomimetics.

As used herein, the term “conservative PAR ligand domain variant” meansa PAR ligand domain that has at least one amino acid substituted byanother amino acid or an amino acid analog that has at least oneproperty similar to that of the original amino acid from the referencePAR ligand domain sequence (Table 3). Examples of properties include,without limitation, similar size, topography, charge, hydrophobicity,hydrophilicity, lipophilicity, covalent-bonding capacity,hydrogen-bonding capacity, a physicochemical property, of the like, orany combination thereof. A conservative PAR ligand domain variant canfunction in substantially the same manner as the reference PAR liganddomain on which the conservative PAR ligand domain variant is based, andcan be substituted for the reference PAR ligand domain in any aspect ofthe present invention. A conservative PAR ligand domain variant maysubstitute one or more amino acids, two or more amino acids, three ormore amino acids, four or more amino acids or five or more amino acidsfrom the reference PAR ligand domain on which the conservative PARligand domain variant is based. A conservative PAR ligand domain variantcan also possess at least 50% amino acid identity, 65% amino acididentity, 75% amino acid identity, 85% amino acid identity or 95% aminoacid identity to the reference PAR ligand domain on which theconservative PAR ligand domain variant is based. Non-limiting examplesof a conservative PAR ligand domain variant include, e.g., conservativePAR1 ligand domain variants, conservative PAR2 ligand domain variants,conservative PAR3 ligand domain variants and conservative PAR4 liganddomain variants.

As used herein, the term “non-conservative PAR ligand domain variant”means a PAR ligand domain in which 1) at least one amino acid is deletedfrom the reference PAR ligand domain on which the non-conservative PARligand domain variant is based; 2) at least one amino acid added to thereference PAR ligand domain on which the non-conservative PAR liganddomain is based; or 3) at least one amino acid is substituted by anotheramino acid or an amino acid analog that does not share any propertysimilar to that of the original amino acid from the reference PAR liganddomain sequence (Table 3). A non-conservative PAR ligand domain variantcan function in substantially the same manner as the reference PARligand domain on which the non-conservative PAR ligand domain is based,and can be substituted for the reference PAR ligand domain in any aspectof the present invention. A non-conservative PAR ligand domain variantcan add one or more amino acids, two or more amino acids, three or moreamino acids, four or more amino acids, five or more amino acids, and tenor more amino acids to the reference PAR ligand domain on which thenon-conservative PAR ligand domain variant is based. A non-conservativePAR ligand domain may substitute one or more amino acids, two or moreamino acids, three or more amino acids, four or more amino acids or fiveor more amino acids from the reference PAR ligand domain on which thenon-conservative PAR ligand domain variant is based. A non-conservativePAR ligand domain variant can also possess at least 50% amino acididentity, 65% amino acid identity, 75% amino acid identity, 85% aminoacid identity or 95% amino acid identity to the reference PAR liganddomain on which the non-conservative PAR ligand domain variant is based.Non-limiting examples of a non-conservative PAR ligand domain variantinclude, e.g., non-conservative PAR1 ligand domain variants,non-conservative PAR2 ligand domain variants, non-conservative PAR3ligand domain variants and non-conservative PAR4 ligand domain variants.

As used herein, the term “PAR ligand domain peptidomimetic” means a PARligand domain that has at least one amino acid substituted by anon-natural oligomer that has at least one property similar to that ofthe first amino acid. Examples of properties include, withoutlimitation, topography of a peptide primary structural element,functionality of a peptide primary structural element, topology of apeptide secondary structural element, functionality of a peptidesecondary structural element, of the like, or any combination thereof. APAR ligand domain peptidomimetic can function in substantially the samemanner as the reference PAR ligand domain on which the PAR ligand domainpeptidomimetic is based, and can be substituted for the reference PARligand domain in any aspect of the present invention. A PAR liganddomain peptidomimetic may substitute one or more amino acids, two ormore amino acids, three or more amino acids, four or more amino acids orfive or more amino acids from the reference PAR ligand domain on whichthe PAR ligand domain peptidomimetic is based. A PAR ligand domainpeptidomimetic can also possess at least 50% amino acid identity, atleast 65% amino acid identity, at least 75% amino acid identity, atleast 85% amino acid identity or at least 95% amino acid identity to thereference PAR ligand domain on which the PAR ligand domainpeptidomimetic is based. For examples of peptidomimetic methods see,e.g., Amy S. Ripka & Daniel H. Rich, Peptidomimetic design, 2 (4) CURR.OPIN. CHEM. BIOL. 441-452 (1998); and M. Angels Estiarte & Daniel H.Rich, Peptidomimetics for Drug Design, 803-861 (BURGER'S MEDICINALCHEMISTRY AND DRUG DISCOVERY Vol. 1 PRINCIPLE AND PRACTICE, Donald J.Abraham ed., Wiley-Interscience, 6^(th) ed 2003). Non-limiting examplesof a PAR ligand domain peptidomimetic include, e.g., PAR1 ligand domainpeptidomimetics, PAR2 ligand domain peptidomimetics, PAR3 ligand domainpeptidomimetics and PAR4 ligand domain peptidomimetics.

Thus, in an embodiment, a PAR ligand domain comprises a naturallyoccurring PAR ligand domain variant, such as, e.g., a PAR ligand domainisoform or a PAR ligand domain subtype. In another embodiment a PARligand domain comprises a non-naturally occurring PAR ligand domainvariant, such as, e.g., a conservative PAR ligand domain variant, anon-conservative PAR ligand domain variant or a PAR ligand domainpeptidomimetic, or any combination thereof.

In another embodiment, a PAR ligand domain comprises a PAR1 liganddomain. In an aspect of this embodiment, a PAR1 ligand domain comprisesSEQ ID NO: 13. In another aspect of this embodiment, a PAR1 liganddomain comprises a naturally occurring PAR1 ligand domain variant, suchas, e.g., a PAR1 ligand domain isoform or a PAR1 ligand domain subtype.In another aspect of this embodiment, a PAR1 ligand domain comprises anaturally occurring PAR1 ligand domain variant of SEQ ID NO: 13, suchas, e.g., a PAR1 ligand domain isoform of SEQ ID NO: 13 or a PAR1 liganddomain subtype of SEQ ID NO: 13. In still another aspect of thisembodiment, a PAR1 ligand domain comprises a non-naturally occurringPAR1 ligand domain variant, such as, e.g., a conservative PAR1 liganddomain variant, a non-conservative PAR1 ligand domain variant or a PAR1ligand domain peptidomimetic, or any combination thereof. In stillanother aspect of this embodiment, a PAR1 ligand domain comprises anon-naturally occurring PAR1 ligand domain variant of SEQ ID NO: 13,such as, e.g., a conservative PAR1 ligand domain variant of SEQ ID NO:13, a non-conservative PAR1 ligand domain variant of SEQ ID NO: 13 or aPAR1 ligand domain peptidomimetic of SEQ ID NO: 13, or any combinationthereof. In other aspects of this embodiment, a PAR1 ligand domaincomprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 133.

In other aspects of this embodiment, a PAR1 ligand domain comprises apolypeptide having, e.g., at least 50% amino acid identity with SEQ IDNO: 13, at least 67% amino acid identity with the SEQ ID NO: 13, or atleast 83% amino acid identity with SEQ ID NO: 13. In still other aspectsof this embodiment, a PAR1 ligand domain comprises a polypeptide having,e.g., at most 50% amino acid identity with SEQ ID NO: 13, at most 67%amino acid identity with the SEQ ID NO: 13, at most 83% amino acididentity with SEQ ID NO: 13.

In other aspects of this embodiment, a PAR1 ligand domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid substitutions relative to SEQ ID NO: 13. In still otheraspects of this embodiment, a PAR1 ligand domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidsubstitutions relative to SEQ ID NO: 13. In yet other aspects of thisembodiment, a PAR1 ligand domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 13. In yetother aspects of this embodiment, a PAR1 ligand domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 13. In still other aspects of this embodiment, a PAR1ligand domain comprises a polypeptide having, e.g., at most one, two orthree non-contiguous amino acid deletions relative to SEQ ID NO: 13. Instill other aspects of this embodiment, a PAR1 ligand domain comprises apolypeptide having, e.g., at least one, two or three non-contiguousamino acid deletions relative to SEQ ID NO: 13.

In other aspects of this embodiment, a PAR1 ligand domain comprises apolypeptide having, e.g., at most two, three or four contiguous aminoacid substitutions relative to SEQ ID NO: 13. In still other aspects ofthis embodiment, a PAR1 ligand domain comprises a polypeptide having,e.g., at least two, three or four contiguous amino acid substitutionsrelative to SEQ ID NO: 13. In yet other aspects of this embodiment, aPAR1 ligand domain comprises a polypeptide having, e.g., at most two,three, four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 13. In yet other aspects of thisembodiment, a PAR1 ligand domain comprises a polypeptide having, e.g.,at least two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 13. In stillother aspects of this embodiment, a PAR1 ligand domain comprises apolypeptide having, e.g., at most two or three contiguous amino aciddeletions relative to SEQ ID NO: 13. In still other aspects of thisembodiment, a PAR1 ligand domain comprises a polypeptide having, e.g.,at least two or three contiguous amino acid deletions relative to SEQ IDNO: 13.

In another embodiment, a PAR ligand domain comprises a PAR2 liganddomain. In an aspect of this embodiment, a PAR2 ligand domain comprisesSEQ ID NO: 24. In another aspect of this embodiment, a PAR2 liganddomain comprises a naturally occurring PAR2 ligand domain variant, suchas, e.g., a PAR2 ligand domain isoform or a PAR2 ligand domain subtype.In another aspect of this embodiment, a PAR2 ligand domain comprises anaturally occurring PAR2 ligand domain variant of SEQ ID NO: 24, suchas, e.g., a PAR2 ligand domain isoform of SEQ ID NO: 24 or a PAR2 liganddomain subtype of SEQ ID NO: 24. In still another aspect of thisembodiment, a PAR2 ligand domain comprises a non-naturally occurringPAR2 ligand domain variant, such as, e.g., a conservative PAR2 liganddomain variant, a non-conservative PAR2 ligand domain variant or a PAR2ligand domain peptidomimetic, or any combination thereof. In stillanother aspect of this embodiment, a PAR2 ligand domain comprises anon-naturally occurring PAR2 ligand domain variant of SEQ ID NO: 24,such as, e.g., a conservative PAR2 ligand domain variant of SEQ ID NO:24, a non-conservative PAR2 ligand domain variant of SEQ ID NO: 24 or aPAR2 ligand domain peptidomimetic of SEQ ID NO: 24, or any combinationthereof. In other aspects of this embodiment, a PAR2 ligand domaincomprises SEQ ID NO: 24 or SEQ ID NO: 25.

In other aspects of this embodiment, a PAR2 ligand domain comprises apolypeptide having, e.g., at least 50% amino acid identity with SEQ IDNO: 24, at least 67% amino acid identity with the SEQ ID NO: 24, or atleast 83% amino acid identity with SEQ ID NO: 24. In still other aspectsof this embodiment, a PAR2 ligand domain comprises a polypeptide having,e.g., at most 50% amino acid identity with SEQ ID NO: 24, at most 67%amino acid identity with the SEQ ID NO: 24, at most 83% amino acididentity with SEQ ID NO: 24.

In other aspects of this embodiment, a PAR2 ligand domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid substitutions relative to SEQ ID NO: 24. In still otheraspects of this embodiment, a PAR2 ligand domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidsubstitutions relative to SEQ ID NO: 24. In yet other aspects of thisembodiment, a PAR2 ligand domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 24. In yetother aspects of this embodiment, a PAR2 ligand domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 24. In still other aspects of this embodiment, a PAR2ligand domain comprises a polypeptide having, e.g., at most one, two orthree non-contiguous amino acid deletions relative to SEQ ID NO: 24. Instill other aspects of this embodiment, a PAR2 ligand domain comprises apolypeptide having, e.g., at least one, two or three non-contiguousamino acid deletions relative to SEQ ID NO: 24.

In other aspects of this embodiment, a PAR2 ligand domain comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid substitutions relative to SEQ ID NO: 24. In still otheraspects of this embodiment, a PAR2 ligand domain comprises a polypeptidehaving, e.g., at least two, three or four contiguous amino acidsubstitutions relative to SEQ ID NO: 24. In yet other aspects of thisembodiment, a PAR2 ligand domain comprises a polypeptide having, e.g.,at most two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 24. In yet otheraspects of this embodiment, a PAR2 ligand domain comprises a polypeptidehaving, e.g., at least two, three, four, five, six, seven, eight, nineor ten contiguous amino acid additions relative to SEQ ID NO: 24. Instill other aspects of this embodiment, a PAR2 ligand domain comprises apolypeptide having, e.g., at most two or three contiguous amino aciddeletions relative to SEQ ID NO: 24. In still other aspects of thisembodiment, a PAR2 ligand domain comprises a polypeptide having, e.g.,at least two or three contiguous amino acid deletions relative to SEQ IDNO: 24.

In another embodiment, a PAR ligand domain comprises a PAR3 liganddomain. In an aspect of this embodiment, a PAR3 ligand domain comprisesSEQ ID NO: 26. In another aspect of this embodiment, a PAR3 liganddomain comprises a naturally occurring PAR3 ligand domain variant, suchas, e.g., a PAR3 ligand domain isoform or a PAR3 ligand domain subtype.In another aspect of this embodiment, a PAR3 ligand domain comprises anaturally occurring PAR3 ligand domain variant of SEQ ID NO: 26, suchas, e.g., a PAR3 ligand domain isoform of SEQ ID NO: 26 or a PAR3 liganddomain subtype of SEQ ID NO: 26. In still another aspect of thisembodiment, a PAR3 ligand domain comprises a non-naturally occurringPAR3 ligand domain variant, such as, e.g., a conservative PAR3 liganddomain variant, a non-conservative PAR3 ligand domain variant or a PAR3ligand domain peptidomimetic, or any combination thereof. In stillanother aspect of this embodiment, a PAR3 ligand domain comprises anon-naturally occurring PAR3 ligand domain variant of SEQ ID NO: 26,such as, e.g., a conservative PAR3 ligand domain variant of SEQ ID NO:26, a non-conservative PAR3 ligand domain variant of SEQ ID NO: 26 or aPAR3 ligand domain peptidomimetic of SEQ ID NO: 26, or any combinationthereof. In other aspects of this embodiment, a PAR3 ligand domaincomprises SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 134.

In other aspects of this embodiment, a PAR3 ligand domain comprises apolypeptide having, e.g., at least 50% amino acid identity with SEQ IDNO: 26, at least 67% amino acid identity with the SEQ ID NO: 26, or atleast 83% amino acid identity with SEQ ID NO: 26. In still other aspectsof this embodiment, a PAR3 ligand domain comprises a polypeptide having,e.g., at most 50% amino acid identity with SEQ ID NO: 26, at most 67%amino acid identity with the SEQ ID NO: 26, at most 83% amino acididentity with SEQ ID NO: 26.

In other aspects of this embodiment, a PAR3 ligand domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid substitutions relative to SEQ ID NO: 26. In still otheraspects of this embodiment, a PAR3 ligand domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidsubstitutions relative to SEQ ID NO: 26. In yet other aspects of thisembodiment, a PAR3 ligand domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 26. In yetother aspects of this embodiment, a PAR3 ligand domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 26. In still other aspects of this embodiment, a PAR3ligand domain comprises a polypeptide having, e.g., at most one, two orthree non-contiguous amino acid deletions relative to SEQ ID NO: 26. Instill other aspects of this embodiment, a PAR3 ligand domain comprises apolypeptide having, e.g., at least one, two or three non-contiguousamino acid deletions relative to SEQ ID NO: 26.

In other aspects of this embodiment, a PAR3 ligand domain comprises apolypeptide having, e.g., at most one, two, three or four contiguousamino acid substitutions relative to SEQ ID NO: 26. In still otheraspects of this embodiment, a PAR3 ligand domain comprises a polypeptidehaving, e.g., at least two, three or four contiguous amino acidsubstitutions relative to SEQ ID NO: 26. In yet other aspects of thisembodiment, a PAR3 ligand domain comprises a polypeptide having, e.g.,at most two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 26. In yet otheraspects of this embodiment, a PAR3 ligand domain comprises a polypeptidehaving, e.g., at least two, three, four, five, six, seven, eight, nineor ten contiguous amino acid additions relative to SEQ ID NO: 26. Instill other aspects of this embodiment, a PAR3 ligand domain comprises apolypeptide having, e.g., at most two or three contiguous amino aciddeletions relative to SEQ ID NO: 26. In still other aspects of thisembodiment, a PAR3 ligand domain comprises a polypeptide having, e.g.,at least two or three contiguous amino acid deletions relative to SEQ IDNO: 26.

In another embodiment, a PAR ligand domain comprises a PAR4 liganddomain. In an aspect of this embodiment, a PAR4 ligand domain comprisesSEQ ID NO: 28. In another aspect of this embodiment, a PAR4 liganddomain comprises a naturally occurring PAR4 ligand domain variant, suchas, e.g., a PAR4 ligand domain isoform or a PAR4 ligand domain subtype.In another aspect of this embodiment, a PAR4 ligand domain comprises anaturally occurring PAR4 ligand domain variant of SEQ ID NO: 28, suchas, e.g., a PAR4 ligand domain isoform of SEQ ID NO: 28 or a PAR4 liganddomain subtype of SEQ ID NO: 28. In still another aspect of thisembodiment, a PAR4 ligand domain comprises a non-naturally occurringPAR4 ligand domain variant, such as, e.g., a conservative PAR4 liganddomain variant, a non-conservative PAR4 ligand domain variant or a PAR4ligand domain peptidomimetic, or any combination thereof. In stillanother aspect of this embodiment, a PAR4 ligand domain comprises anon-naturally occurring PAR4 ligand domain variant of SEQ ID NO: 28,such as, e.g., a conservative PAR4 ligand domain variant of SEQ ID NO:28, a non-conservative PAR4 ligand domain variant of SEQ ID NO: 28 or aPAR4 ligand domain peptidomimetic of SEQ ID NO: 28, or any combinationthereof. In other aspects of this embodiment, a PAR4 ligand domaincomprises SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ IDNO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQID NO: 46, SEQ ID NO: 47, SEQ ID NO: 135 or SEQ ID NO: 160.

In other aspects of this embodiment, a PAR4 ligand domain comprises apolypeptide having, e.g., at least 50% amino acid identity with SEQ IDNO: 28, at least 67% amino acid identity with the SEQ ID NO: 28, or atleast 83% amino acid identity with SEQ ID NO: 28. In still other aspectsof this embodiment, a PAR4 ligand domain comprises a polypeptide having,e.g., at most 50% amino acid identity with SEQ ID NO: 28, at most 67%amino acid identity with the SEQ ID NO: 28, at most 83% amino acididentity with SEQ ID NO: 28.

In other aspects of this embodiment, a PAR4 ligand domain comprises apolypeptide having, e.g., at most one, two, three or four non-contiguousamino acid substitutions relative to SEQ ID NO: 28. In still otheraspects of this embodiment, a PAR4 ligand domain comprises a polypeptidehaving, e.g., at least one, two, three or four non-contiguous amino acidsubstitutions relative to SEQ ID NO: 28. In yet other aspects of thisembodiment, a PAR4 ligand domain comprises a polypeptide having, e.g.,at most one, two, three, four, five, six, seven, eight, nine or tennon-contiguous amino acid additions relative to SEQ ID NO: 28. In yetother aspects of this embodiment, a PAR4 ligand domain comprises apolypeptide having, e.g., at least one, two, three, four, five, six,seven, eight, nine or ten non-contiguous amino acid additions relativeto SEQ ID NO: 28. In still other aspects of this embodiment, a PAR4ligand domain comprises a polypeptide having, e.g., at most one, two orthree non-contiguous amino acid deletions relative to SEQ ID NO: 28. Instill other aspects of this embodiment, a PAR4 ligand domain comprises apolypeptide having, e.g., at least one, two or three non-contiguousamino acid deletions relative to SEQ ID NO: 28.

In other aspects of this embodiment, a PAR4 ligand domain comprises apolypeptide having, e.g., at most two, three or four contiguous aminoacid substitutions relative to SEQ ID NO: 28. In still other aspects ofthis embodiment, a PAR4 ligand domain comprises a polypeptide having,e.g., at least two, three or four contiguous amino acid substitutionsrelative to SEQ ID NO: 28. In yet other aspects of this embodiment, aPAR4 ligand domain comprises a polypeptide having, e.g., at most two,three, four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 28. In yet other aspects of thisembodiment, a PAR4 ligand domain comprises a polypeptide having, e.g.,at least two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 28. In stillother aspects of this embodiment, a PAR4 ligand domain comprises apolypeptide having, e.g., at most two or three contiguous amino aciddeletions relative to SEQ ID NO: 28. In still other aspects of thisembodiment, a PAR4 ligand domain comprises a polypeptide having, e.g.,at least two or three contiguous amino acid deletions relative to SEQ IDNO: 28.

When a PAR protease cleaves the extracellular amino-terminus of a PAR, anew amino acid terminus is generated that functions as a tetheredligand. Currently it is believed that the amino terminus location of thetethered ligand is critical for the ligand to effectively bind to thesecond extracellular loop region of the receptor that comprises theligand binding domain. It is envisioned that a modified Clostridialtoxin of the present specification can comprise a PAR ligand domain inany and all locations with the proviso that formation of the di-chainmolecule will result in the free amino terminus of the PAR liganddomain. Non-limiting examples include, locating the PAR ligand domain atthe amino terminus of the Clostridial toxin enzymatic domain; locatingthe PAR ligand domain at the amino terminus of the Clostridial toxintranslocation domain; and locating the PAR ligand domain at the aminoterminus of the Clostridial toxin binding domain (FIG. 4).

Thus, in an embodiment, a modified Clostridial toxin comprises a PARligand domain; a Clostridial toxin enzymatic domain; a Clostridial toxintranslocation domain; and a Clostridial toxin binding domain; whereinthe PAR ligand domain is located at the amino terminus of theClostridial toxin enzymatic domain. In an aspect of this embodiment, thePAR ligand domain can be located at the amino terminus of the enzymaticdomain when the amino to carboxyl linear organization of the Clostridialtoxin single chain molecule is enzymatic domain, translocation domainand binding domain. In another aspect of this embodiment, the PAR liganddomain can be located at the amino terminus of the enzymatic domain whenthe amino to carboxyl linear organization of the Clostridial toxinsingle chain molecule is enzymatic domain, binding domain andtranslocation domain.

In another embodiment, a modified Clostridial toxin comprises a PARligand domain; a Clostridial toxin enzymatic domain; a Clostridial toxintranslocation domain; and a Clostridial toxin binding domain; whereinthe PAR ligand domain is located at the amino terminus of theClostridial toxin translocation domain. In an aspect of this embodiment,the PAR ligand domain can also be located at the amino terminus of thetranslocation domain when the amino to carboxyl linear organization ofthe Clostridial toxin single chain molecule is binding domain, enzymaticdomain and translocation domain. In another aspect of this embodiment,the PAR ligand domain can also be located at the amino terminus of thetranslocation domain when the amino to carboxyl linear organization ofthe Clostridial toxin single chain molecule is enzymatic domain,translocation domain and binding domain.

In still another embodiment, a modified Clostridial toxin comprises aPAR ligand domain; a Clostridial toxin enzymatic domain; a Clostridialtoxin translocation domain; and a Clostridial toxin binding domain;wherein the PAR ligand domain is located at the amino terminus of theClostridial toxin binding domain. In an aspect of this embodiment, thePAR ligand domain can also be located at the amino terminus of thebinding domain when the amino to carboxyl linear organization of theClostridial toxin single chain molecule is enzymatic domain, bindingdomain and translocation domain.

TABLE 4 Amino Terminus Region SEQ ID Light Chain Toxin NO:PAR Ligand Domain Region BoNT/A 1 M-PAR Ligand DomainPFVNKQFNYKDPVNGVDIA BoNT/B 2 M-PAR Ligand Domain PVTINNFNYNDPIDNNNIIBoNT/C1 3 M-PAR Ligand Domain PITINNFNYSDPVDNKNIL BoNT/D 4M-PAR Ligand Domain TWPVKDFNYSDPVNDNDIL BoNT/E 5 M-PAR Ligand DomainPKINSFNYNDPVNDRTILY BoNT/F 6 M-PAR Ligand Domain PVAINSFNYNDPVNDDTILBoNT/G 7 M-PAR Ligand Domain PVNIKXFNYNDPINNDDII TeNT 8M-PAR Ligand Domain PITINNFRYSDPVNNDTII The amino acid sequencedisplayed are as follows: BoNT/A, residues 2-20 of SEQ ID No: 1; BoNT/B,residues 2-20 of SEQ ID No: 2; BoNT/C1, residues v of SEQ ID No: 3;BoNT/D, residues 2-20 of SEQ ID No: 4; BoNT/E, residues 2-20 of SEQ IDNo: 5; BoNT/F, residues 2-20 of SEQ ID No: 6; BoNT/G, residues 2-20 ofSEQ ID No: 7; and TeNT, residues 2-20 of SEQ ID No: 8.

In yet another embodiment, the location of the PAR ligand domain islocated at the amino terminus of the modified Clostridial toxin. In sucha location, the PAR ligand domain can bind to a ligand binding domain ofa PAR; proteolytic cleavage is not necessary to unmask the PAR liganddomain. As used herein, the term “unmask” means that the amino terminusof a PAR ligand domain is free to bind to a ligand binding domain of aPAR. It is known in the art that when adding a polypeptide that isoperationally-linked to the amino terminus of another polypeptidecomprising the start methionine that this methionine residue can bedeleted (Table 4). This is due to the fact that the added polypeptidewill contain a new start methionine and that the original startmethionine may reduce optimal expression of the fusion protein.

In yet another embodiment, the location of the PAR ligand domain is notlocated at the amino terminus of the modified Clostridial toxin. In sucha location, the PAR ligand domain can not bind to a ligand bindingdomain of a PAR. The PAR ligand domain is considered masked because itis necessary to unmask a PAR ligand domain so that this domain can bindto a ligand binding domain of a PAR. As used herein, the term “masked”means that the amino terminus of a PAR ligand domain is unable to bindto the ligand binding domain of a PAR. To unmask a PAR ligand domain ofa modified Clostridial toxin, a protease cleavage site can be placed infront of the PAR ligand domain in such a manner that, upon cleavage withan appropriate protease, the masked PAR ligand domain becomes unmaskedand is now capable of binding a PAR ligand binding domain. It isenvisioned that any and all proteases that can cleave a modifiedClostridial toxin disclosed in the present specification so as to unmaska PAR ligand domain can be used, including without limitation, aClostridial toxin protease cleavage site found in the di-chain loop, aPAR protease cleavage site used to unmask the tethered ligand in vivo,and an exogenous protease cleavage site.

As mentioned above, a Clostridial toxin is converted from a singlepolypeptide form into a di-chain molecule by proteolytic cleavage. Whilethe identity of the protease is currently unknown, the di-chain loopprotease cleavage site for many Clostridial toxins has been determined.In BoNTs, cleavage at K448-A449 converts the single polypeptide form ofBoNT/A into the di-chain form; cleavage at K441-A442 converts the singlepolypeptide form of BoNT/B into the di-chain form; cleavage at K449-T450converts the single polypeptide form of BoNT/C1 into the di-chain form;cleavage at R445-D446 converts the single polypeptide form of BoNT/Dinto the di-chain form; cleavage at R422-K423 converts the singlepolypeptide form of BoNT/E into the di-chain form; cleavage at K439-A440converts the single polypeptide form of BoNT/F into the di-chain form;and cleavage at K446-5447 converts the single polypeptide form of BoNT/Ginto the di-chain form. Proteolytic cleavage of the single polypeptideform of TeNT at A457-5458 results in the di-chain form. Such a di-chainloop protease cleavage site is operably-linked in-frame to a modifiedClostridial toxin as a fusion protein. However, it should also be notedthat additional cleavage sites within the di-chain look also appear tobe cleaved resulting in the generation of a small peptide fragment beinglost. As a non-limiting example, BoNT/A single-chain polypeptide cleaveultimately results in the loss of a ten amino acid fragment within thedi-chain loop.

Thus, in an embodiment, proteolytic cleavage of an endogenousClostridial toxin di-chain loop protease cleavage site is used to unmaska PAR ligand domain. In aspects of this embodiment, a PAR ligand domainis unmasked by proteolytic cleavage of, e.g., a BoNT/A di-chain loopprotease cleavage site, a BoNT/B di-chain loop protease cleavage site, aBoNT/C1 di-chain loop protease cleavage site, a BoNT/D di-chain loopprotease cleavage site, a BoNT/E di-chain loop protease cleavage site, aBoNT/F di-chain loop protease cleavage site, a BoNT/G di-chain loopprotease cleavage site or a TeNT di-chain loop protease cleavage site.

A wide variety of endogenous PAR proteases are known to cleave a PAR insuch a manner as to unmask the tethered ligand and, therefore, can alsobe used to unmask the PAR ligand domain. The coagulant protease Thrombinis the physiological activator of PAR1, PAR3 and PAR4. Other PARproteases, however, can also activate PAR receptors by proteolyticcleavage including, without limitation, APC, Cathepsin G, Factor VIIa,Factor Xa, Granzyme A, Gingipains-R, Plasmin and Trypsins (Table 2).PAR2 can also be activated by multiple proteases including, withoutlimitation, Acrosien, Der P1, Der P3, Der P9, Factor VIIa, Factor Xa,Gingipains-R, MT-SP1, Proteinase-3, Trypsins and Tryptases (Table 2). Itis envisioned that both endogenous protease cleavage sites foundassociated with a particular PAR ligand domain, as well as exogenousprotease cleavage sites from other PAR ligand domains can be used tocleave a modified Clostridial toxin disclosed in the presentspecification in order to unmask the PAR ligand binding domain. Such aPAR protease cleavage site is operably-linked in-frame to a modifiedClostridial toxin as a fusion protein. As a non-limiting example, a PAR1ligand domain can be unmasked using the protease cleavage siteassociated with the in vivo PAR1 molecule, or a PAR1 ligand domain canbe unmasked using the protease cleavage site associated with PAR2, PAR3or PAR4 (Table 2). As another non-limiting example, a PAR2 ligand domaincan be unmasked using the protease cleavage site associated with the invivo PAR2 molecule, or a PAR2 ligand domain can be unmasked using theprotease cleavage site associated PAR1, PAR3 or PAR4 (Table 2). As stillanother non-limiting example, a PAR3 ligand domain can be unmasked usingthe protease cleavage site associated with the in vivo PAR3 molecule, ora PAR3 ligand domain can be unmasked using the protease cleavage siteassociated with PAR1, PAR2 or PAR4 (Table 2). As yet anothernon-limiting example, a PAR4 ligand domain can be unmasked using theprotease cleavage site associated with the in vivo PAR4 molecule, or aPAR4 ligand domain can be unmasked using the protease cleavage siteassociated with PAR1, PAR2 or PAR3 (Table 2).

Thus, in an embodiment, proteolytic cleavage of an endogenous PAR1protease cleavage site is used to unmask a PAR ligand domain. In aspectsof this embodiment, a PAR ligand domain is unmasked by proteolyticcleavage of, e.g., an APC protease cleavage site, a Factor Xa proteasecleavage site, a Granzyme A protease cleavage site, a Gingipains-Rprotease cleavage site, a Thrombin protease cleavage site or a Trypsinprotease cleavage site. In other aspects of this embodiment, a PAR1protease cleavage site is cleaved by, e.g., an APC protease, a Factor Xaprotease, a Granzyme A protease, a Gingipains-R protease, a Thrombinprotease or a Trypsin protease.

In another embodiment, proteolytic cleavage of an endogenous PAR2protease cleavage site is used to unmask a PAR ligand domain. In aspectsof this embodiment, a PAR ligand domain is unmasked by proteolyticcleavage of, e.g., an Acrosien protease cleavage site, a Der P1 proteasecleavage site, a Der P3 protease cleavage site, a Der P9 proteasecleavage site, a Factor VIIa protease cleavage site, a Factor Xaprotease cleavage site, a Gingipains-R protease cleavage site, a MT-SP1protease cleavage site, a Proteinase-3 protease cleavage site, a Trypsinprotease cleavage site or a Tryptase protease cleavage site. In otheraspects of this embodiment, a PAR2 protease cleavage site is cleaved by,e.g., an Acrosien protease, a Der P1 protease, a Der P3 protease, a DerP9 protease, a Factor VIIa protease, a Factor Xa protease, aGingipains-R protease, a MT-SP1 protease, a Proteinase-3 protease, aTrypsin protease or a Tryptase protease.

In another embodiment, proteolytic cleavage of an endogenous PAR3protease cleavage site is used to unmask a PAR ligand domain. In anaspect of this embodiment, a PAR ligand domain is unmasked byproteolytic cleavage of, e.g., a Thrombin protease cleavage site. Inanother aspect of this embodiment, a PAR3 protease cleavage site iscleaved by, e.g., a Thrombin protease.

In another embodiment, proteolytic cleavage of an endogenous PAR4protease cleavage site is used to unmask a PAR ligand domain. In aspectsof this embodiment, a PAR ligand domain is unmasked by proteolyticcleavage of, e.g., a Cathepsin G protease cleavage site, a Factor VIIaprotease cleavage site, a Factor Xa protease cleavage site, aGingipains-R protease cleavage site, a Plasmin protease cleavage site, aThrombin protease cleavage site or a Trypsin protease cleavage site. Inother aspects of this embodiment, a PAR4 protease cleavage site iscleaved by, e.g., a Cathepsin G protease, a Factor VIIa protease, aFactor Xa protease, a Gingipains-R protease, a Plasmin protease, aThrombin protease or a Trypsin protease.

It is also envisioned that an exogenous protease cleavage site can beused to unmask a PAR ligand domain. Such an exogenous protease cleavagesite is operably-linked in-frame to a modified Clostridial toxin as afusion protein. Non-limiting examples of protease cleavage sitesinclude, e.g., an enterokinase cleavage site (Table 5); a Thrombincleavage site (Table 5); a Factor Xa cleavage site (Table 5); a humanrhinovirus 3C protease cleavage site (Table 4); a tobacco etch virus(TEV) protease cleavage site (Table 5); a dipeptidyl aminopeptidasecleavage site and a small ubiquitin-like modifier (SUMO)/ubiquitin-likeprotein-1(ULP-1) protease cleavage site, such as, e.g., MADSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID. NO: 67). As a non-limiting example, a PAR1 liganddomain can be unmasked using a bovine enterokinase protease cleavagesite, a Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3Cprotease cleavage site, a SUMO/ULP-1 protease cleavage site, a Thrombinprotease cleavage site or a Factor Xa protease cleavage site (Table 5).As another non-limiting example, a PAR2 ligand domain can be unmaskedusing a bovine enterokinase protease cleavage site, a Tobacco Etch Virusprotease cleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site ora Factor Xa protease cleavage site (Table 5). As still anothernon-limiting example, a PAR3 ligand domain can be unmasked using abovine enterokinase protease cleavage site, a Tobacco Etch Virusprotease cleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site ora Factor Xa protease cleavage site (Table 5). As yet anothernon-limiting example, a PAR4 ligand domain can be unmasked using abovine enterokinase protease cleavage site, a Tobacco Etch Virusprotease cleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site ora Factor Xa protease cleavage site (Table 5).

Thus, in an embodiment, proteolytic cleavage of an exogenous proteasecleavage site is used to unmask a PAR ligand domain. In aspects of thisembodiment, a PAR ligand domain is unmasked by proteolytic cleavage of,e.g., a bovine enterokinase protease cleavage site, a Tobacco Etch Virusprotease cleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site ora Factor Xa protease cleavage site. In other aspects of this embodiment,a PAR protease cleavage site is cleaved by, e.g., a bovine enterokinaseprotease, a Tobacco Etch Virus protease, a Human Rhinovirus 3C protease,a SUMO/ULP-1 protease, a Thrombin protease or a Factor Xa protease,thereby unmasking a PAR ligand domain.

TABLE 5 Exogenous Protease Cleavage Sites Protease Cleavage Non-limitingSEQ ID Site Consensus Sequence Examples NO: Bovine enterokinase DDDDK*DDDDK* 50 Tobacco Etch Virus E P⁵ P⁴YP²Q*(G/S) ENLYFQ*G 51 (TEV)where P², P⁴ and P⁵ can be any amino acid ENLYFQ*S 52 ENIYTQ*G 53ENIYTQ*S 54 ENIYLQ*G 55 ENIYLQ*S 56 ENVYFQ*G 57 ENVYSQ*S 58 ENVYSQ*G 59ENVYSQ*S 60 Human Rhinovirus 3C P⁵P⁴LFQ*GP EALFQ*GP 61P⁴ is G, A, V, L, I, M, S or T and P⁵ can any EVLFQ*GP 62amino acid, with D or E preferred. ELLFQ*GP 63 DALFQ*GP 64 DVLFQ*GP 65DLLFQ*GP 66 SUMO/ULP-1 Tertiary structure polypeptide-G* 67 ThrombinP³P²(R/K)*P^(1′), GVR*G 68where P³ is any amino acid and P² or P^(1′) is G SAR*G 69with the other position being any amino acid SLR*G 70 DGR*I 71 QGK*I 72Thrombin P⁴P³P(R/K)*P^(1′)P^(2′) LVPR*GS 73where P^(1′) and P^(2′) can be any amino acid except LVPK*GS 74for acidic amino acids like D or E; and P³ FIPR*TF 75and P⁴ are hydrophobic amino acids like F, L, VLPR*SF 76I, Y, W, V, M, P, C or A IVPR*SF 77 IVPR*GY 78 VVPR*GV 79 VLPR*LI 80VMPR*SL 81 MFPR*SL 82 Coagulation Factor Xa I(E/D)GR* IDGR* 83 IEGR* 84An asterisks (*) indicates the peptide bond that is cleaved by theindicated protease.

In another embodiment, proteolytic cleavage of an exogenous proteasecleavage site is used to unmask a PAR1 ligand domain. In aspects of thisembodiment, a PAR1 ligand domain is unmasked by proteolytic cleavage of,e.g., a bovine enterokinase protease cleavage site, a Tobacco Etch Virusprotease cleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site ora Factor Xa protease cleavage site. In other aspects of this embodiment,a PAR1 protease cleavage site is cleaved by, e.g., a bovine enterokinaseprotease, a Tobacco Etch Virus protease, a Human Rhinovirus 3C protease,a SUMO/ULP-1 protease, a Thrombin protease or a Factor Xa protease,thereby unmasking a PAR1 ligand domain.

In another embodiment, proteolytic cleavage of an exogenous proteasecleavage site is used to unmask a PAR2 ligand domain. In aspects of thisembodiment, a PAR2 ligand domain is unmasked by proteolytic cleavage of,e.g., a bovine enterokinase protease cleavage site, a Tobacco Etch Virusprotease cleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site ora Factor Xa protease cleavage site. In other aspects of this embodiment,a PAR2 protease cleavage site is cleaved by, e.g., a bovine enterokinaseprotease, a Tobacco Etch Virus protease, a Human Rhinovirus 3C protease,a SUMO/ULP-1 protease, a Thrombin protease or a Factor Xa protease,thereby unmasking a PAR2 ligand domain.

In still another embodiment, proteolytic cleavage of an exogenousprotease cleavage site is used to unmask a PAR3 ligand domain. Inaspects of this embodiment, a PAR3 ligand domain is unmasked byproteolytic cleavage of, e.g., a bovine enterokinase protease cleavagesite, a Tobacco Etch Virus protease cleavage site, a Human Rhinovirus 3Cprotease cleavage site, a SUMO/ULP-1 protease cleavage site, a Thrombinprotease cleavage site or a Factor Xa protease cleavage site. In otheraspects of this embodiment, a PAR3 protease cleavage site is cleaved by,e.g., a bovine enterokinase protease, a Tobacco Etch Virus protease, aHuman Rhinovirus 3C protease, a SUMO/ULP-1 protease, a Thrombin proteaseor a Factor Xa protease, thereby unmasking a PAR3 ligand domain.

In another embodiment, proteolytic cleavage of an exogenous proteasecleavage site is used to unmask a PAR4 ligand domain. In aspects of thisembodiment, a PAR4 ligand domain is unmasked by proteolytic cleavage of,e.g., a bovine enterokinase protease cleavage site, a Tobacco Etch Virusprotease cleavage site, a Human Rhinovirus 3C protease cleavage site, aSUMO/ULP-1 protease cleavage site, a Thrombin protease cleavage site ora Factor Xa protease cleavage site. In other aspects of this embodiment,a PAR4 protease cleavage site is cleaved by, e.g., a bovine enterokinaseprotease, a Tobacco Etch Virus protease, a Human Rhinovirus 3C protease,a SUMO/ULP-1 protease, a Thrombin protease or a Factor Xa protease,thereby unmasking a PAR4 ligand domain.

It is understood that a modified Clostridial toxin disclosed in thepresent specification can optionally include one or more additionalcomponents. As a non-limiting example of an optional component, amodified Clostridial toxin can further comprise a flexible regioncomprising a flexible spacer. Non-limiting examples of a flexible spacerinclude, e.g., a G-spacer GGGGS (SEQ ID NO: 48) or an A-spacer EAAAK(SEQ ID NO: 49). A flexible region comprising flexible spacers can beused to adjust the length of a polypeptide region in order to optimize acharacteristic, attribute or property of a polypeptide. Such a flexibleregion is operably-linked in-frame to the modified Clostridial toxin asa fusion protein. As a non-limiting example, a polypeptide regioncomprising one or more flexible spacers in tandem can be use to betterexpose a protease cleavage site thereby facilitating cleavage of thatsite by a protease. As another non-limiting example, a polypeptideregion comprising one or more flexible spacers in tandem can be use tobetter present a ligand domain, thereby facilitating the binding of thatligand domain to its binding domain on a receptor.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise a flexible region comprisinga flexible spacer. In another embodiment, a modified Clostridial toxindisclosed in the present specification can further comprise flexibleregion comprising a plurality of flexible spacers in tandem. In aspectsof this embodiment, a flexible region can comprise in tandem, e.g., atleast 1 G-spacer, at least 2 G-spacers, at least 3 G-spacers, at least 4G-spacers or at least 5 G-spacers. In other aspects of this embodiment,a flexible region can comprise in tandem, e.g., at most 1 G-spacer, atmost 2 G-spacers, at most 3 G-spacers, at most 4 G-spacers or at most 5G-spacers. In still other aspects of this embodiment, a flexible regioncan comprise in tandem, e.g., at least 1 A-spacer, at least 2 A-spacers,at least 3 A-spacers, at least 4 A-spacers or at least 5 A-spacers. Instill other aspects of this embodiment, a flexible region can comprisein tandem, e.g., at most 1 A-spacer, at most 2 A-spacers, at most 3A-spacers, at most 4 A-spacers or at most 5 A-spacers. In another aspectof this embodiment, a modified Clostridial toxin can comprise a flexibleregion comprising one or more copies of the same flexible spacers, oneor more copies of different flexible-spacer regions, or any combinationthereof.

As another non-limiting example of an optional component, a modifiedClostridial toxin can further comprise an epitope-binding region. Anepitope-binding region can be used in a wide variety of proceduresinvolving, e.g., protein purification and protein visualization. Such anepitope-binding region is operably-linked in-frame to a modifiedClostridial toxin as a fusion protein. Non-limiting examples of anepitope-binding region include, e.g., FLAG, Express™, human Influenzavirus hemagluttinin (HA), human p62^(c-Myc) protein (c-MYC), VesicularStomatitis Virus Glycoprotein (VSV-G), glycoprotein-D precursor ofHerpes simplex virus (HSV), V5, and AU1; affinity-binding, such as.e.g., polyhistidine (HIS), streptavidin binding peptide (strep), andbiotin or a biotinylation sequence; peptide-binding regions, such as.e.g., the glutathione binding domain of glutathione-S-transferase, thecalmodulin binding domain of the calmodulin binding protein, and themaltose binding domain of the maltose binding protein. Non-limitingexamples of specific protocols for selecting, making and using anappropriate binding peptide are described in, e.g., Epitope Tagging, pp.17.90-17.93 (Sambrook and Russell, eds., Molecular Cloning A LaboratoryManual, Vol. 3, 3^(rd) ed. 2001); Antibodies: A Laboratory Manual(Edward Harlow & David Lane, eds., Cold Spring Harbor Laboratory Press,2^(nd) ed. 1998); and Using Antibodies: A Laboratory Manual: PortableProtocol No. I (Edward Harlow & David Lane, Cold Spring HarborLaboratory Press, 1998). In addition, non-limiting examples of bindingpeptides as well as well-characterized reagents, conditions andprotocols are readily available from commercial vendors that include,without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BDBiosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif.These protocols are routine procedures well within the scope of oneskilled in the art and from the teaching herein.

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise an epitope-binding region. Inanother embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprises a plurality ofepitope-binding regions. In aspects of this embodiment, a modifiedClostridial toxin can comprise, e.g., at least 1 epitope-binding region,at least 2 epitope-binding regions, at least 3 epitope-binding regions,at least 4 epitope-binding regions or at least 5 epitope-bindingregions. In other aspects of this embodiment, a modified Clostridialtoxin can comprise, e.g., at most 1 epitope-binding region, at most 2epitope-binding regions, at most 3 epitope-binding regions, at most 4epitope-binding regions or at most 5 epitope-binding regions. In anotheraspect of this embodiment, a modified Clostridial toxin can comprise oneor more copies of the same epitope-binding region, one or more copies ofdifferent epitope-binding regions, or any combination thereof. Thelocation of an epitope-binding region can be in various positions,including, without limitation, at the amino terminus of a modifiedClostridial toxin, within a modified Clostridial toxin, or at thecarboxyl terminus of a modified Clostridial toxin.

As still another non-limiting example of an optional component, amodified Clostridial toxin can further comprise an exogenous proteasecleavage site. An exogenous protease cleavage site can be used in a widevariety of procedures involving, e.g., removal of an epitope-bindingregion by proteolytic cleavage, conversion of a Clostridial toxin singlechain polypeptide into the di-chain form or, as mentioned above,unmasking of a PAR ligand domain. Such an exogenous protease cleavagesite is operably-linked in-frame to a modified Clostridial toxin as afusion protein. Non-limiting examples of protease cleavage sitesinclude, e.g., an enterokinase cleavage site (Table 5); a Thrombincleavage site (Table 5); a Factor Xa cleavage site (Table 5); a humanrhinovirus 3C protease cleavage site (Table 4); a tobacco etch virus(TEV) protease cleavage site (Table 5); a dipeptidyl aminopeptidasecleavage site and a small ubiquitin-like modifier (SUMO)/ubiquitin-likeprotein-1(ULP-1) protease cleavage site, such as, e.g.,MADSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID. NO: 67).

Thus, in an embodiment, a modified Clostridial toxin disclosed in thepresent specification can further comprise an exogenous proteasecleavage site. In another embodiment, a modified Clostridial toxindisclosed in the present specification can further comprises a pluralityof exogenous protease cleavage sites. In aspects of this embodiment, amodified Clostridial toxin can comprise, e.g., at least 1 exogenousprotease cleavage site, at least 2 exogenous protease cleavage sites, atleast 3 exogenous protease cleavage sites, at least 4 exogenous proteasecleavage sites or at least 5 exogenous protease cleavage sites. In otheraspects of this embodiment, a modified Clostridial toxin can comprise,e.g., at most 1 exogenous protease cleavage site, at most 2 exogenousprotease cleavage sites, at most 3 exogenous protease cleavage sites, atmost 4 exogenous protease cleavage sites or at most 5 exogenous proteasecleavage sites. In another aspect of this embodiment, a modifiedClostridial toxin can comprise one or more copies of the same exogenousprotease cleavage site, one or more copies of different exogenousprotease cleavage sites, or any combination thereof.

The location of an exogenous protease cleavage site may be in a varietyof positions, including, without limitation, between an epitope-bindingregion and a modified Clostridial toxin in order to facilitate removalof the epitope-binding region by proteolytic cleavage or within thedi-chain loop of the modified Clostridial toxin in order to facilitatethe conversion of the single-chain polypeptide form of the toxin intothe di-chain form.

It is envisioned that an exogenous protease cleavage site can be used toremove an epitope-binding region. As mentioned above, epitope bindingregions can be used in protein purification procedures and it is oftendesirable to remove such epitope binding regions after the protein ispurified. A common way of doing so is to have a protease cleavage sitein between the protein of interest and the epitope binding region,whereby proteolytic cleavage of the protease cleavage site separates theprotein of interest from the epitope binding region. Non-limitingexamples of protease cleavage sites used for the removal ofepitope-binding regions as well as well-characterized proteases,reagents, conditions and protocols are readily available from commercialvendors that include, without limitation, BD Biosciences-Clontech, PaloAlto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen,Inc, Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene,La Jolla, Calif. The selection, making and use of an appropriateprotease cleavage site are routine procedures within the scope of oneskilled in the art and from the teaching herein.

Thus, in an embodiment, an exogenous protease cleavage site is locatedbetween an epitope-binding peptide and a modified Clostridial toxin. Inother aspects of this embodiment, a bovine enterokinase cleavage site islocated between an epitope-binding region and a modified Clostridialtoxin, a Tobacco Etch Virus protease cleavage site is located between anepitope-binding region and a modified Clostridial toxin, a HumanRhinovirus 3C protease cleavage site is located between anepitope-binding region and a modified Clostridial toxin, a SUMO/ULP-1protease cleavage site is located between an epitope-binding region anda modified Clostridial toxin, a Thrombin protease cleavage site islocated between an epitope-binding region and a modified Clostridialtoxin, or a Coagulation Factor Xa protease cleavage site is locatedbetween an epitope-binding region and a modified Clostridial toxin. Inother aspects of the embodiment, the bovine enterokinase proteasecleavage site located between an epitope-binding region and a modifiedClostridial toxin comprises SEQ ID NO: 50. In other aspects of theembodiment, the Tobacco Etch Virus protease cleavage site locatedbetween an epitope-binding region and a modified Clostridial toxincomprises SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:59 or SEQ ID NO: 60. In still other aspects of the embodiment, the HumanRhinovirus 3C protease cleavage site located between an epitope-bindingregion and a modified Clostridial toxin comprises SEQ ID NO: 61, SEQ IDNO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66. Inyet other aspects of the embodiment, the SUMO/ULP-1 protease cleavagesite located between an epitope-binding region and a modifiedClostridial toxin comprises SEQ ID NO: 67. In further other aspects ofthe embodiment, the Thrombin protease cleavage site located between anepitope-binding region and a modified Clostridial toxin comprises SEQ IDNO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO:82. In other aspects of the embodiment, the Coagulation Factor Xaprotease cleavage site located between an epitope-binding region and amodified Clostridial toxin comprises SEQ ID NO: 83 or SEQ ID NO: 84.

TABLE 6 Di-chain Loop Region SEQ ID Light ChainDi-chain Loop Protease Cleavage Site Heavy Chain Toxin NO: Region RegionRegion BoNT/A 1 NMNFTKLKNFTGLFEFYKLL CVRGIITSKTKSLDKGYNK*----ALNDLCIKVNNWDL BoNT/B 2  KQAYEEISKEHLAVYKIQM CKSVK*-------------------APGICIDVDNEDL BoNT/C1 3   PALRKVNPENMLYLFTKF CHKAIDGRSLYNK*------------TLDCRELLVKNTDL BoNT/D 4   PALQKLSSESVVDLFTKV CLRLTKNSR*---------------DDSTCIKVKNNRL BoNT/E 5    IITPITGRGLVKKIIRF CKNIVSVKGIR*--------------KSICIEINNGEL BoNT/F 6    IIDSIPDKGLVEKIVKF CKSVIPRKGTK*------------APPRLCIRVNNSEL BoNT/G 7  KEAYEEISLEHLVIYRIAM CKPVMYKNTGK*--------------SEQCIIVNNEDL TeNT 8  TNAFRNVDGSGLVSKLIGL CKKIIPPTNIRENLYNRTA*SLTDLGGELCIKIKNEDL The amino acid sequence displayed are as follows: BoNT/A,residues 325-462 of SEQ ID No: 1; BoNT/B, residues 332-454 of SEQ ID No:2; BoNT/C1, residues 334-463 of SEQ ID No: 3; BoNT/D, residues 334-458of SEQ ID No: 4; BoNT/E, residues 311-434 of SEQ ID No: 5; BoNT/F,residues 328-453 of SEQ ID No: 6; BoNT/G, residues 331-458 of SEQ ID No:7; and TeNT, residues 334-474 of SEQ ID No: 8. An asterisks (*)indicates the peptide bond that is cleaved by a Clostridial toxinprotease.

It is envisioned that an exogenous protease cleavage site can be used toconvert the single-chain polypeptide form of a modified Clostridialtoxin disclosed in the present specification into the di-chain form. Asmentioned above, Clostridial toxins are translated as a single-chainpolypeptide of approximately 150 kDa that is subsequently cleaved byproteolytic scission within a disulfide loop by a naturally-occurringprotease. This posttranslational processing yields a di-chain moleculecomprising an approximately 50 kDa light chain (LC) and an approximately100 kDa heavy chain (HC) held together by a single disulphide bond andnoncovalent interactions. While the naturally-occurring protease iscurrently not known, cleavage occurs within the di-chain loop regionbetween the two cysteine residues that form the disulfide bridge (Table6). Replacement of the naturally-occurring protease cleavage site withan exogenous protease cleavage site will enable cleavage of a modifiedClostridial toxin disclosed in the present specification when expressedin an organism that does not produce the endogenous Clostridial proteaseused to cleave the di-chain loop region of a toxin.

Thus in an embodiment, an exogenous protease cleavage site is locatedwithin the di-chain loop of a modified Clostridial toxin. In aspects ofthis embodiment, a bovine enterokinase cleavage site is located withinthe di-chain loop of a modified Clostridial toxin, a Tobacco Etch Virusprotease cleavage site is located within the di-chain loop of a modifiedClostridial toxin, a Human Rhinovirus 3C protease cleavage site islocated within the di-chain loop of a modified Clostridial toxin, aSUMO/ULP-1 protease cleavage site is located within the di-chain loop ofa modified Clostridial toxin, a Thrombin protease cleavage site islocated within the di-chain loop of a modified Clostridial toxin, or aCoagulation Factor Xa protease cleavage site is located within thedi-chain loop of a modified Clostridial toxin. In other aspects of theembodiment, the bovine enterokinase protease cleavage site locatedwithin the di-chain loop of a modified Clostridial toxin comprises SEQID NO: 50. In other aspects of the embodiment, the Tobacco Etch Virusprotease cleavage site located within the di-chain loop of a modifiedClostridial toxin comprises SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO: 59 or SEQ ID NO: 60. In still other aspects of theembodiment, the Human Rhinovirus 3C protease cleavage site locatedwithin the di-chain loop of a modified Clostridial toxin comprises SEQID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 orSEQ ID NO: 66. In yet other aspects of the embodiment, the SUMO/ULP-1protease cleavage site located within the di-chain loop of a modifiedClostridial toxin comprises SEQ ID NO: 67. In further other aspects ofthe embodiment, the Thrombin protease cleavage site located within thedi-chain loop of a modified Clostridial toxin comprises SEQ ID NO: 68,SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ IDNO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82. Inother aspects of the embodiment, the Coagulation Factor Xa proteasecleavage site located within the di-chain loop of a modified Clostridialtoxin comprises SEQ ID NO: 83 or SEQ ID NO: 84.

Aspects of the present invention provide, in part modified Clostridialtoxins. Non-limiting examples of Clostridial toxin modificationsdisclosed in the present specification include, e.g., addition of a PARligand domain, addition of a protease cleavage site, rearrangement ofthe enzymatic, translocation and binding domains, addition of a spacerregion and addition of an epitope-binding region. It is understood thatall such modifications do not substantially affect the ability of aClostridial toxin to intoxicate a cell. As used herein, the term “do notsubstantially affect” means a Clostridial toxin can still execute theoverall cellular mechanism whereby a Clostridial toxin enters a neuronand inhibits neurotransmitter release and encompasses the binding of aClostridial toxin to a low or high affinity receptor complex, theinternalization of the toxin/receptor complex, the translocation of theClostridial toxin light chain into the cytoplasm and the enzymaticmodification of a Clostridial toxin substrate. In aspects of thisembodiment, the modified Clostridial toxin is, e.g., at least 10% astoxic as a naturally-occurring Clostridial toxin, at least 20% as toxicas a naturally-occurring Clostridial toxin, at least 30% as toxic as anaturally-occurring Clostridial toxin, at least 40% as toxic as anaturally-occurring Clostridial toxin, at least 50% as toxic as anaturally-occurring Clostridial toxin, at least 60% as toxic as anaturally-occurring Clostridial toxin, at least 70% as toxic as anaturally-occurring Clostridial toxin, at least 80% as toxic as anaturally-occurring Clostridial toxin, at least 90% as toxic as anaturally-occurring Clostridial toxin or at least 95% as toxic as anaturally-occurring Clostridial toxin. In aspects of this embodiment,the modified Clostridial toxin is, e.g., at most 10% as toxic as anaturally-occurring Clostridial toxin, at most 20% as toxic as anaturally-occurring Clostridial toxin, at most 30% as toxic as anaturally-occurring Clostridial toxin, at most 40% as toxic as anaturally-occurring Clostridial toxin, at most 50% as toxic as anaturally-occurring Clostridial toxin, at most 60% as toxic as anaturally-occurring Clostridial toxin, at most 70% as toxic as anaturally-occurring Clostridial toxin, at most 80% as toxic as anaturally-occurring Clostridial toxin, at most 90% as toxic as anaturally-occurring Clostridial toxin or at most 95% as toxic as anaturally-occurring Clostridial toxin.

Aspects of the present invention provide, in part polynucleotidemolecules. As used herein, the term “polynucleotide molecule” issynonymous with “nucleic acid molecule” and means a polymeric form ofnucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides, ofany length. It is envisioned that any and all polynucleotide moleculesthat can encode a modified Clostridial toxin disclosed in the presentspecification can be useful, including, without limitationnaturally-occurring and non-naturally-occurring DNA molecules andnaturally-occurring and non-naturally-occurring RNA molecules.Non-limiting examples of naturally-occurring and non-naturally-occurringDNA molecules include single-stranded DNA molecules, double-stranded DNAmolecules, genomic DNA molecules, cDNA molecules, vector constructs,such as, e.g., plasmid constructs, phagmid constructs, bacteriophageconstructs, retroviral constructs and artificial chromosome constructs.Non-limiting examples of naturally-occurring and non-naturally-occurringRNA molecules include single-stranded RNA, double stranded RNA and mRNA.

Thus, in an embodiment, a polynucleotide molecule encodes a Clostridialtoxin comprises a Clostridial toxin enzymatic domain, a Clostridialtoxin translocation domain and a Clostridial toxin binding domain. In anaspect of this embodiment, a polynucleotide molecule encodes aClostridial toxin comprises a naturally occurring Clostridial toxinvariant, such as, e.g., a Clostridial toxin isoform or a Clostridialtoxin subtype. In another aspect of this embodiment, a polynucleotidemolecule encodes a Clostridial toxin comprises a non-naturally occurringClostridial toxin variant, such as, e.g., a conservative Clostridialtoxin variant, a non-conservative Clostridial toxin variant or an activeClostridial toxin fragment, or any combination thereof. In anotheraspect of this embodiment, a polynucleotide molecule encodes aClostridial toxin comprises a Clostridial toxin enzymatic domain or anactive fragment thereof, a Clostridial toxin translocation domain or anactive fragment thereof, a Clostridial toxin binding domain or an activefragment thereof, or any combination thereof. In other aspects of thisembodiment, a Clostridial toxins comprises a BoNT/A, a BoNT/B, aBoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G or a TeNT.

In another embodiment, a polynucleotide molecule encodes a Clostridialtoxin comprising a BoNT/A. In an aspect of this embodiment, apolynucleotide molecule encodes a BoNT/A comprising a BoNT/A enzymaticdomain, a BoNT/A translocation domain and a BoNT/A binding domain. Inanother aspect of this embodiment, a polynucleotide molecule encodes aBoNT/A comprising SEQ ID NO: 1. In another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/A comprising a naturallyoccurring BoNT/A variant, such as, e.g., a BoNT/A isoform or a BoNT/Asubtype. In another aspect of this embodiment, a polynucleotide moleculeencodes a BoNT/A comprising a naturally occurring BoNT/A variant of SEQID NO: 1, such as, e.g., a BoNT/A isoform of SEQ ID NO: 1 or a BoNT/Asubtype of SEQ ID NO: 1. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/A comprising a non-naturallyoccurring BoNT/A variant, such as, e.g., a conservative BoNT/A variant,a non-conservative BoNT/A variant or an active BoNT/A fragment, or anycombination thereof. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/A comprising a non-naturallyoccurring BoNT/A variant of SEQ ID NO: 1, such as, e.g., a conservativeBoNT/A variant of SEQ ID NO: 1, a non-conservative BoNT/A variant of SEQID NO: 1 or an active BoNT/A fragment of SEQ ID NO: 1, or anycombination thereof. In yet another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/A comprising a BoNT/A enzymaticdomain or an active fragment thereof, a BoNT/A translocation domain oran active fragment thereof, a BoNT/A binding domain or an activefragment thereof, or any combination thereof. In yet another aspect ofthis embodiment, a BoNT/A comprising a BoNT/A enzymatic domain of aminoacids 1-448 from SEQ ID NO: 1 or an active fragment thereof, a BoNT/Atranslocation domain of amino acids 449-860 from SEQ ID NO: 1 or anactive fragment thereof, a BoNT/A binding domain of amino acids 861-1296from SEQ ID NO: 1 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/A comprising a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 1, at least 75% amino acid identity with theSEQ ID NO: 1, at least 80% amino acid identity with SEQ ID NO: 1, atleast 85% amino acid identity with SEQ ID NO: 1, at least 90% amino acididentity with SEQ ID NO: 1 or at least 95% amino acid identity with SEQID NO: 1. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a BoNT/A comprising a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 1, at most 75% amino acididentity with the SEQ ID NO: 1, at most 80% amino acid identity with SEQID NO: 1, at most 85% amino acid identity with SEQ ID NO: 1, at most 90%amino acid identity with SEQ ID NO: 1 or at most 95% amino acid identitywith SEQ ID NO: 1.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/A comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 1. Inother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/A comprising a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 1. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/A comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 1. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Acomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 1. In stillother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/A comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 1. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Acomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 1.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/A comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 1. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Acomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 1. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/A comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 1. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Acomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 1. In still otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Acomprising a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 1. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Acomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 1.

In another embodiment, a polynucleotide molecule encodes a Clostridialtoxin comprising a BoNT/B. In an aspect of this embodiment, apolynucleotide molecule encodes a BoNT/B comprising a BoNT/B enzymaticdomain, a BoNT/B translocation domain and a BoNT/B binding domain. Inanother aspect of this embodiment, a polynucleotide molecule encodes aBoNT/B comprising SEQ ID NO: 2. In another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/B comprising a naturallyoccurring BoNT/B variant, such as, e.g., a BoNT/B isoform or a BoNT/Bsubtype. In another aspect of this embodiment, a polynucleotide moleculeencodes a BoNT/B comprising a naturally occurring BoNT/B variant of SEQID NO: 2, such as, e.g., a BoNT/B isoform of SEQ ID NO: 2 or a BoNT/Bsubtype of SEQ ID NO: 2. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/B comprising a non-naturallyoccurring BoNT/B variant, such as, e.g., a conservative BoNT/B variant,a non-conservative BoNT/B variant or an active BoNT/B fragment, or anycombination thereof. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/B comprising a non-naturallyoccurring BoNT/B variant of SEQ ID NO: 2, such as, e.g., a conservativeBoNT/B variant of SEQ ID NO: 2, a non-conservative BoNT/B variant of SEQID NO: 2 or an active BoNT/B fragment of SEQ ID NO: 2, or anycombination thereof. In yet another aspect of this embodiment, a BoNT/Bcomprising a BoNT/B enzymatic domain or an active fragment thereof, aBoNT/B translocation domain or active fragment thereof, a BoNT/B bindingdomain or active fragment thereof, and any combination thereof. In yetanother aspect of this embodiment, a BoNT/B comprising a BoNT/Benzymatic domain of amino acids 1-441 from SEQ ID NO: 2 or activefragment thereof, a BoNT/B translocation domain of amino acids 442-847from SEQ ID NO: 2 or active fragment thereof, a BoNT/B binding domain ofamino acids 848-1291 from SEQ ID NO: 2 or active fragment thereof, andany combination thereof.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/B comprising a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 2, at least 75% amino acid identity with theSEQ ID NO: 2, at least 80% amino acid identity with SEQ ID NO: 2, atleast 85% amino acid identity with SEQ ID NO: 2, at least 90% amino acididentity with SEQ ID NO: 2 or at least 95% amino acid identity with SEQID NO: 2. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a BoNT/B comprising a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 2, at most 75% amino acididentity with the SEQ ID NO: 2, at most 80% amino acid identity with SEQID NO: 2, at most 85% amino acid identity with SEQ ID NO: 2, at most 90%amino acid identity with SEQ ID NO: 2 or at most 95% amino acid identitywith SEQ ID NO: 2.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/B comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 2. Inother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/B comprising a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 2. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/B comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 2. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Bcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 2. In stillother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/B comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 2. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Bcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 2.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/B comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 2. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Bcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 2. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/B comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 2. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Bcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 2. In still otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Bcomprising a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 2. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Bcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 2.

In another embodiment, a polynucleotide molecule encodes a Clostridialtoxin comprising a BoNT/C1. In an aspect of this embodiment, apolynucleotide molecule encodes a BoNT/C1 comprising a BoNT/C1 enzymaticdomain, a BoNT/C1 translocation domain and a BoNT/C1 binding domain. Inanother aspect of this embodiment, a polynucleotide molecule encodes aBoNT/C1 comprising SEQ ID NO: 3. In another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/C1 comprising a naturallyoccurring BoNT/C1 variant, such as, e.g., a BoNT/C1 isoform or a BoNT/C1subtype. In another aspect of this embodiment, a polynucleotide moleculeencodes a BoNT/C1 comprising a naturally occurring BoNT/C1 variant ofSEQ ID NO: 3, such as, e.g., a BoNT/C1 isoform of SEQ ID NO: 3 or aBoNT/C1 subtype of SEQ ID NO: 3. In still another aspect of thisembodiment, a polynucleotide molecule encodes a BoNT/C1 comprising anon-naturally occurring BoNT/C1 variant, such as, e.g., a conservativeBoNT/C1 variant, a non-conservative BoNT/C1 variant or an active BoNT/C1fragment, or any combination thereof. In still another aspect of thisembodiment, a polynucleotide molecule encodes a BoNT/C1 comprising anon-naturally occurring BoNT/C1 variant of SEQ ID NO: 3, such as, e.g.,a conservative BoNT/C1 variant of SEQ ID NO: 3, a non-conservativeBoNT/C1 variant of SEQ ID NO: 3 or an active BoNT/C1 fragment of SEQ IDNO: 3, or any combination thereof. In yet another aspect of thisembodiment, a polynucleotide molecule encodes a BoNT/C1 comprising aBoNT/C1 enzymatic domain or active fragment thereof, a BoNT/C1translocation domain or active fragment thereof, a BoNT/C1 bindingdomain or active fragment thereof, and any combination thereof. In yetanother aspect of this embodiment, a polynucleotide molecule encodes aBoNT/C1 comprising a BoNT/C1 enzymatic domain of amino acid 1-449 fromSEQ ID NO: 3 or active fragment thereof, a BoNT/C1 translocation domainof amino acids 450-855 from SEQ ID NO: 3 or active fragment thereof, aBoNT/C1 binding domain of amino acids 856-1291 from SEQ ID NO: 3 oractive fragment thereof, and any combination thereof.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/C1 comprising a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 3, at least 75% amino acid identity with theSEQ ID NO: 3, at least 80% amino acid identity with SEQ ID NO: 3, atleast 85% amino acid identity with SEQ ID NO: 3, at least 90% amino acididentity with SEQ ID NO: 3 or at least 95% amino acid identity with SEQID NO: 3. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a BoNT/C1 comprising a polypeptide having, e.g., atmost 70% amino acid identity with SEQ ID NO: 3, at most 75% amino acididentity with the SEQ ID NO: 3, at most 80% amino acid identity with SEQID NO: 3, at most 85% amino acid identity with SEQ ID NO: 3, at most 90%amino acid identity with SEQ ID NO: 3 or at most 95% amino acid identitywith SEQ ID NO: 3.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/C1 comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 3. Inother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/C1 comprising a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 3. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/C1 comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 3. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/C1comprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 3. In stillother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/C1 comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 3. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/C1comprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 3.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/C1 comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 3. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/C1comprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 3. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/C1 comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 3. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/C1comprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 3. In still otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/C1comprising a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 3. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/C1comprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 3.

In another embodiment, a polynucleotide molecule encodes a Clostridialtoxin comprising a BoNT/D. In an aspect of this embodiment, apolynucleotide molecule encodes a BoNT/D comprising a BoNT/D enzymaticdomain, a BoNT/D translocation domain and a BoNT/D binding domain. Inanother aspect of this embodiment, a polynucleotide molecule encodes aBoNT/D comprising SEQ ID NO: 4. In another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/D comprising a naturallyoccurring BoNT/D variant, such as, e.g., a BoNT/D isoform or a BoNT/Dsubtype. In another aspect of this embodiment, a polynucleotide moleculeencodes a BoNT/D comprising a naturally occurring BoNT/D variant of SEQID NO: 4, such as, e.g., a BoNT/D isoform of SEQ ID NO: 4 or a BoNT/Dsubtype of SEQ ID NO: 4. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/D comprising a non-naturallyoccurring BoNT/D variant, such as, e.g., a conservative BoNT/D variant,a non-conservative BoNT/D variant or an active BoNT/D fragment, or anycombination thereof. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/D comprising a non-naturallyoccurring BoNT/D variant of SEQ ID NO: 4, such as, e.g., a conservativeBoNT/D variant of SEQ ID NO: 4, a non-conservative BoNT/D variant of SEQID NO: 4 or an active BoNT/D fragment of SEQ ID NO: 4, or anycombination thereof. In yet another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/D comprising a BoNT/D enzymaticdomain or an active fragment thereof, a BoNT/D translocation domain oran active fragment thereof, a BoNT/D binding domain or an activefragment thereof, or any combination thereof. In yet another aspect ofthis embodiment, a BoNT/D comprising a BoNT/D enzymatic domain of aminoacids 1-442 from SEQ ID NO: 4 or an active fragment thereof, a BoNT/Dtranslocation domain of amino acids 443-851 from SEQ ID NO: 4 or anactive fragment thereof, a BoNT/D binding domain of amino acids 852-1276from SEQ ID NO: 4 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/D comprising a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 4, at least 75% amino acid identity with theSEQ ID NO: 4, at least 80% amino acid identity with SEQ ID NO: 4, atleast 85% amino acid identity with SEQ ID NO: 4, at least 90% amino acididentity with SEQ ID NO: 4 or at least 95% amino acid identity with SEQID NO: 4. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a BoNT/D comprising a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 4, at most 75% amino acididentity with the SEQ ID NO: 4, at most 80% amino acid identity with SEQID NO: 4, at most 85% amino acid identity with SEQ ID NO: 4, at most 90%amino acid identity with SEQ ID NO: 4 or at most 95% amino acid identitywith SEQ ID NO: 4.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/D comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 4. Inother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/D comprising a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 4. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/D comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 4. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Dcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 4. In stillother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/D comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 4. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Dcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 4.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/D comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 4. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Dcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 4. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/D comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 4. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Dcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 4. In still otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Dcomprising a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 4. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Dcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 4.

In another embodiment, a polynucleotide molecule encodes a Clostridialtoxin comprising a BoNT/E. In an aspect of this embodiment, apolynucleotide molecule encodes a BoNT/E comprising a BoNT/E enzymaticdomain, a BoNT/E translocation domain and a BoNT/E binding domain. Inanother aspect of this embodiment, a polynucleotide molecule encodes aBoNT/E comprising SEQ ID NO: 5. In another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/E comprising a naturallyoccurring BoNT/E variant, such as, e.g., a BoNT/E isoform or a BoNT/Esubtype. In another aspect of this embodiment, a polynucleotide moleculeencodes a BoNT/E comprising a naturally occurring BoNT/E variant of SEQID NO: 5, such as, e.g., a BoNT/E isoform of SEQ ID NO: 5 or a BoNT/Esubtype of SEQ ID NO: 5. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/E comprising a non-naturallyoccurring BoNT/E variant, such as, e.g., a conservative BoNT/E variant,a non-conservative BoNT/E variant or an active BoNT/E fragment, or anycombination thereof. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/E comprising a non-naturallyoccurring BoNT/E variant of SEQ ID NO: 5, such as, e.g., a conservativeBoNT/E variant of SEQ ID NO: 5, a non-conservative BoNT/E variant of SEQID NO: 5 or an active BoNT/E fragment of SEQ ID NO: 5, or anycombination thereof. In yet another aspect of this embodiment, a BoNT/Ecomprising a BoNT/E enzymatic domain or an active fragment thereof, aBoNT/E translocation domain or active fragment thereof, a BoNT/E bindingdomain or active fragment thereof, and any combination thereof. In yetanother aspect of this embodiment, a BoNT/E comprising a BoNT/Eenzymatic domain of amino acids 1-422 from SEQ ID NO: 5 or activefragment thereof, a BoNT/E translocation domain of amino acids 423-834from SEQ ID NO: 5 or active fragment thereof, a BoNT/E binding domain ofamino acids 835-1252 from SEQ ID NO: 5 or active fragment thereof, andany combination thereof.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/E comprising a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 5, at least 75% amino acid identity with theSEQ ID NO: 5, at least 80% amino acid identity with SEQ ID NO: 5, atleast 85% amino acid identity with SEQ ID NO: 5, at least 90% amino acididentity with SEQ ID NO: 5 or at least 95% amino acid identity with SEQID NO: 5. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a BoNT/E comprising a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 5, at most 75% amino acididentity with the SEQ ID NO: 5, at most 80% amino acid identity with SEQID NO: 5, at most 85% amino acid identity with SEQ ID NO: 5, at most 90%amino acid identity with SEQ ID NO: 5 or at most 95% amino acid identitywith SEQ ID NO: 5.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/E comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 5. Inother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/E comprising a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 5. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/E comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 5. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Ecomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 5. In stillother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/E comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 5. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Ecomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 5.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/E comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 5. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Ecomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 5. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/E comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 5. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Ecomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 5. In still otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Ecomprising a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 5. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Ecomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 5.

In another embodiment, a polynucleotide molecule encodes a Clostridialtoxin comprising a BoNT/F. In an aspect of this embodiment, apolynucleotide molecule encodes a BoNT/F comprising a BoNT/F enzymaticdomain, a BoNT/F translocation domain and a BoNT/F binding domain. Inanother aspect of this embodiment, a polynucleotide molecule encodes aBoNT/F comprising SEQ ID NO: 6. In another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/F comprising a naturallyoccurring BoNT/F variant, such as, e.g., a BoNT/F isoform or a BoNT/Fsubtype. In another aspect of this embodiment, a polynucleotide moleculeencodes a BoNT/F comprising a naturally occurring BoNT/F variant of SEQID NO: 6, such as, e.g., a BoNT/F isoform of SEQ ID NO: 6 or a BoNT/Fsubtype of SEQ ID NO: 6. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/F comprising a non-naturallyoccurring BoNT/F variant, such as, e.g., a conservative BoNT/F variant,a non-conservative BoNT/F variant or an active BoNT/F fragment, or anycombination thereof. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/F comprising a non-naturallyoccurring BoNT/F variant of SEQ ID NO: 6, such as, e.g., a conservativeBoNT/F variant of SEQ ID NO: 6, a non-conservative BoNT/F variant of SEQID NO: 6 or an active BoNT/F fragment of SEQ ID NO: 6, or anycombination thereof. In yet another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/F comprising a BoNT/F enzymaticdomain or active fragment thereof, a BoNT/F translocation domain oractive fragment thereof, a BoNT/F binding domain or active fragmentthereof, and any combination thereof. In yet another aspect of thisembodiment, a polynucleotide molecule encodes a BoNT/F comprising aBoNT/F enzymatic domain of amino acid 1-436 from SEQ ID NO: 6 or activefragment thereof, a BoNT/F translocation domain of amino acids 437-852from SEQ ID NO: 6 or active fragment thereof, a BoNT/F binding domain ofamino acids 853-1274 from SEQ ID NO: 6 or active fragment thereof, andany combination thereof.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/F comprising a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 6, at least 75% amino acid identity with theSEQ ID NO: 6, at least 80% amino acid identity with SEQ ID NO: 6, atleast 85% amino acid identity with SEQ ID NO: 6, at least 90% amino acididentity with SEQ ID NO: 6 or at least 95% amino acid identity with SEQID NO: 6. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a BoNT/F comprising a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 6, at most 75% amino acididentity with the SEQ ID NO: 6, at most 80% amino acid identity with SEQID NO: 6, at most 85% amino acid identity with SEQ ID NO: 6, at most 90%amino acid identity with SEQ ID NO: 6 or at most 95% amino acid identitywith SEQ ID NO: 6.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/F comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 6. Inother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/F comprising a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 6. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/F comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 6. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Fcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 6. In stillother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/F comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 6. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Fcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 6.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/F comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 6. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Fcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 6. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/F comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 6. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Fcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 6. In still otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Fcomprising a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 6. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Fcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 6.

In another embodiment, a polynucleotide molecule encodes a Clostridialtoxin comprising a BoNT/G. In an aspect of this embodiment, apolynucleotide molecule encodes a BoNT/G comprising a BoNT/G enzymaticdomain, a BoNT/G translocation domain and a BoNT/G binding domain. Inanother aspect of this embodiment, a polynucleotide molecule encodes aBoNT/G comprising SEQ ID NO: 7. In another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/G comprising a naturallyoccurring BoNT/G variant, such as, e.g., a BoNT/G isoform or a BoNT/Gsubtype. In another aspect of this embodiment, a polynucleotide moleculeencodes a BoNT/G comprising a naturally occurring BoNT/G variant of SEQID NO: 7, such as, e.g., a BoNT/G isoform of SEQ ID NO: 7 or a BoNT/Gsubtype of SEQ ID NO: 7. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/G comprising a non-naturallyoccurring BoNT/G variant, such as, e.g., a conservative BoNT/G variant,a non-conservative BoNT/G variant or an active BoNT/G fragment, or anycombination thereof. In still another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/D comprising a non-naturallyoccurring BoNT/G variant of SEQ ID NO: 7, such as, e.g., a conservativeBoNT/G variant of SEQ ID NO: 7, a non-conservative BoNT/G variant of SEQID NO: 7 or an active BoNT/G fragment of SEQ ID NO: 7, or anycombination thereof. In yet another aspect of this embodiment, apolynucleotide molecule encodes a BoNT/G comprising a BoNT/G enzymaticdomain or an active fragment thereof, a BoNT/G translocation domain oran active fragment thereof, a BoNT/G binding domain or an activefragment thereof, or any combination thereof. In yet another aspect ofthis embodiment, a BoNT/G comprising a BoNT/G enzymatic domain of aminoacids 1-442 from SEQ ID NO: 7 or an active fragment thereof, a BoNT/Gtranslocation domain of amino acids 443-852 from SEQ ID NO: 7 or anactive fragment thereof, a BoNT/G binding domain of amino acids 853-1297from SEQ ID NO: 7 or an active fragment thereof, and any combinationthereof.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/G comprising a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 7, at least 75% amino acid identity with theSEQ ID NO: 7, at least 80% amino acid identity with SEQ ID NO: 7, atleast 85% amino acid identity with SEQ ID NO: 7, at least 90% amino acididentity with SEQ ID NO: 7 or at least 95% amino acid identity with SEQID NO: 7. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a BoNT/G comprising a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 7, at most 75% amino acididentity with the SEQ ID NO: 7, at most 80% amino acid identity with SEQID NO: 7, at most 85% amino acid identity with SEQ ID NO: 7, at most 90%amino acid identity with SEQ ID NO: 7 or at most 95% amino acid identitywith SEQ ID NO: 7.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/G comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 7. Inother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/G comprising a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 7. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/G comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 7. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Gcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 7. In stillother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/G comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 7. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Gcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 7.

In other aspects of this embodiment, a polynucleotide molecule encodes aBoNT/G comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 7. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Gcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 7. In yetother aspects of this embodiment, a polynucleotide molecule encodes aBoNT/G comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 7. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Gcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 7. In still otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Gcomprising a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 7. In otheraspects of this embodiment, a polynucleotide molecule encodes a BoNT/Gcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 7.

In another embodiment, a polynucleotide molecule encodes a Clostridialtoxin comprising a TeNT. In an aspect of this embodiment, apolynucleotide molecule encodes a TeNT comprising a TeNT enzymaticdomain, a TeNT translocation domain and a TeNT binding domain. In anaspect of this embodiment, a polynucleotide molecule encodes a TeNTcomprising SEQ ID NO: 8. In another aspect of this embodiment, apolynucleotide molecule encodes a TeNT comprising a naturally occurringTeNT variant, such as, e.g., a TeNT isoform or a TeNT subtype. Inanother aspect of this embodiment, a polynucleotide molecule encodes aTeNT comprising a naturally occurring TeNT variant of SEQ ID NO: 8, suchas, e.g., a TeNT isoform of SEQ ID NO: 8 or a TeNT subtype of SEQ ID NO:8. In still another aspect of this embodiment, a polynucleotide moleculeencodes a TeNT comprising a non-naturally occurring TeNT variant, suchas, e.g., a conservative TeNT variant, a non-conservative TeNT variantor an active TeNT fragment, or any combination thereof. In still anotheraspect of this embodiment, a polynucleotide molecule encodes a TeNTcomprising a non-naturally occurring TeNT variant of SEQ ID NO: 8, suchas, e.g., a conservative TeNT variant of SEQ ID NO: 8, anon-conservative TeNT variant of SEQ ID NO: 8 or an active TeNT fragmentof SEQ ID NO: 8, or any combination thereof. In yet another aspect ofthis embodiment, a TeNT comprising a TeNT enzymatic domain or an activefragment thereof, a TeNT translocation domain or active fragmentthereof, a TeNT binding domain or active fragment thereof, and anycombination thereof. In yet another aspect of this embodiment, a TeNTcomprising a TeNT enzymatic domain of amino acids 1-441 from SEQ ID NO:8 or active fragment thereof, a TeNT translocation domain of amino acids442-870 from SEQ ID NO: 8 or active fragment thereof, a TeNT bindingdomain of amino acids 871-1315 from SEQ ID NO: 8 or active fragmentthereof, and any combination thereof.

In other aspects of this embodiment, a polynucleotide molecule encodes aTeNT comprising a polypeptide having, e.g., at least 70% amino acididentity with SEQ ID NO: 8, at least 75% amino acid identity with theSEQ ID NO: 8, at least 80% amino acid identity with SEQ ID NO: 8, atleast 85% amino acid identity with SEQ ID NO: 8, at least 90% amino acididentity with SEQ ID NO: 8 or at least 95% amino acid identity with SEQID NO: 8. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a TeNT comprising a polypeptide having, e.g., at most70% amino acid identity with SEQ ID NO: 8, at most 75% amino acididentity with the SEQ ID NO: 8, at most 80% amino acid identity with SEQID NO: 8, at most 85% amino acid identity with SEQ ID NO: 8, at most 90%amino acid identity with SEQ ID NO: 8 or at most 95% amino acid identitywith SEQ ID NO: 8.

In other aspects of this embodiment, a polynucleotide molecule encodes aTeNT comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 8. Inother aspects of this embodiment, a polynucleotide molecule encodes aTeNT comprising a polypeptide having, e.g., at least one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid substitutions relative to SEQ ID NO: 8. In yetother aspects of this embodiment, a polynucleotide molecule encodes aTeNT comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 8. In otheraspects of this embodiment, a polynucleotide molecule encodes a TeNTcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid deletions relative to SEQ ID NO: 8. In stillother aspects of this embodiment, a polynucleotide molecule encodes aTeNT comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 8. In otheraspects of this embodiment, a polynucleotide molecule encodes a TeNTcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500non-contiguous amino acid additions relative to SEQ ID NO: 8.

In other aspects of this embodiment, a polynucleotide molecule encodes aTeNT comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 8. In otheraspects of this embodiment, a polynucleotide molecule encodes a TeNTcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid substitutions relative to SEQ ID NO: 8. In yetother aspects of this embodiment, a polynucleotide molecule encodes aTeNT comprising a polypeptide having, e.g., at most one, two, three,four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 8. In otheraspects of this embodiment, a polynucleotide molecule encodes a TeNTcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid deletions relative to SEQ ID NO: 8. In still otheraspects of this embodiment, a polynucleotide molecule encodes a TeNTcomprising a polypeptide having, e.g., at most one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 8. In otheraspects of this embodiment, a polynucleotide molecule encodes a TeNTcomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500contiguous amino acid additions relative to SEQ ID NO: 8.

In still another embodiment, a polynucleotide molecule encodes a PARligand domain comprising a naturally occurring PAR ligand domainvariant, such as, e.g., a PAR ligand domain isoform or a PAR liganddomain subtype. In another embodiment, a polynucleotide molecule encodesa PAR ligand domain comprising a non-naturally occurring PAR liganddomain variant, such as, e.g., a conservative PAR ligand domain variant,a non-conservative PAR ligand domain variant or a PAR ligand domainpeptidomimetic, or any combination thereof.

In still another embodiment, a polynucleotide molecule encodes a PARligand domain comprising a PAR1 ligand domain. In an aspect of thisembodiment, a polynucleotide molecule encodes a PAR1 ligand domaincomprising SEQ ID NO: 13. In another aspect of this embodiment, apolynucleotide molecule encodes a PAR1 ligand domain comprising anaturally occurring PAR1 ligand domain variant, such as, e.g., a PAR1ligand domain isoform or a PAR1 ligand domain subtype. In another aspectof this embodiment, a polynucleotide molecule encodes a PAR1 liganddomain comprising a naturally occurring PAR1 ligand domain variant ofSEQ ID NO: 13, such as, e.g., a PAR1 ligand domain isoform of SEQ ID NO:13 or a PAR1 ligand domain subtype of SEQ ID NO: 13. In still anotheraspect of this embodiment, a polynucleotide molecule encodes a PAR1ligand domain comprising a non-naturally occurring PAR1 ligand domainvariant, such as, e.g., a conservative PAR1 ligand domain variant, anon-conservative PAR1 ligand domain variant or a PAR1 ligand domainpeptidomimetic, or any combination thereof. In still another aspect ofthis embodiment, a polynucleotide molecule encodes a PAR1 ligand domaincomprising a non-naturally occurring PAR1 ligand domain variant of SEQID NO: 13, such as, e.g., a conservative PAR1 ligand domain variant ofSEQ ID NO: 13, a non-conservative PAR1 ligand domain variant of SEQ IDNO: 13 or a PAR1 ligand domain peptidomimetic of SEQ ID NO: 13, or anycombination thereof. In other aspects of this embodiment, apolynucleotide molecule encodes a PAR1 ligand domain comprising SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 orSEQ ID NO: 23.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR1 ligand domain comprising a polypeptide having, e.g., at least 50%amino acid identity with SEQ ID NO: 13, at least 67% amino acid identitywith the SEQ ID NO: 13, or at least 83% amino acid identity with SEQ IDNO: 13. In still other aspects of this embodiment, a polynucleotidemolecule encodes a PAR1 ligand domain comprising a polypeptide having,e.g., at most 50% amino acid identity with SEQ ID NO: 13, at most 67%amino acid identity with the SEQ ID NO: 13, at most 83% amino acididentity with SEQ ID NO: 13.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR1 ligand domain comprising a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid substitutions relative toSEQ ID NO: 13. In still other aspects of this embodiment, apolynucleotide molecule encodes a PAR1 ligand domain comprising apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to SEQ ID NO: 13. Inyet other aspects of this embodiment, a polynucleotide molecule encodesa PAR1 ligand domain comprising a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine or ten non-contiguousamino acid additions relative to SEQ ID NO: 13. In yet other aspects ofthis embodiment, a polynucleotide molecule encodes a PAR1 ligand domaincomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acid additionsrelative to SEQ ID NO: 13. In still other aspects of this embodiment, apolynucleotide molecule encodes a PAR1 ligand domain comprising apolypeptide having, e.g., at most one, two or three non-contiguous aminoacid deletions relative to SEQ ID NO: 13. In still other aspects of thisembodiment, a polynucleotide molecule encodes a PAR1 ligand domaincomprising a polypeptide having, e.g., at least one, two or threenon-contiguous amino acid deletions relative to SEQ ID NO: 13.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR1 ligand domain comprising a polypeptide having, e.g., at most two,three or four contiguous amino acid substitutions relative to SEQ ID NO:13. In still other aspects of this embodiment, a polynucleotide moleculeencodes a PAR1 ligand domain comprising a polypeptide having, e.g., atleast two, three or four contiguous amino acid substitutions relative toSEQ ID NO: 13. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a PAR1 ligand domain comprising a polypeptide having,e.g., at most two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 13. In yet otheraspects of this embodiment, a polynucleotide molecule encodes a PAR1ligand domain comprising a polypeptide having, e.g., at least two,three, four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 13. In still other aspects of thisembodiment, a polynucleotide molecule encodes a PAR1 ligand domaincomprising a polypeptide having, e.g., at most two or three contiguousamino acid deletions relative to SEQ ID NO: 13. In still other aspectsof this embodiment, a polynucleotide molecule encodes a PAR1 liganddomain comprising a polypeptide having, e.g., at least two or threecontiguous amino acid deletions relative to SEQ ID NO: 13.

In still another embodiment, a polynucleotide molecule encodes a PARligand domain comprising a PAR2 ligand domain. In an aspect of thisembodiment, a polynucleotide molecule encodes a PAR2 ligand domaincomprising SEQ ID NO: 24. In another aspect of this embodiment, apolynucleotide molecule encodes a PAR2 ligand domain comprising anaturally occurring PAR2 ligand domain variant, such as, e.g., a PAR2ligand domain isoform or a PAR2 ligand domain subtype. In another aspectof this embodiment, a polynucleotide molecule encodes a PAR2 liganddomain comprising a naturally occurring PAR2 ligand domain variant ofSEQ ID NO: 24, such as, e.g., a PAR2 ligand domain isoform of SEQ ID NO:24 or a PAR2 ligand domain subtype of SEQ ID NO: 24. In still anotheraspect of this embodiment, a polynucleotide molecule encodes a PAR2ligand domain comprising a non-naturally occurring PAR2 ligand domainvariant, such as, e.g., a conservative PAR2 ligand domain variant, anon-conservative PAR2 ligand domain variant or a PAR2 ligand domainpeptidomimetic, or any combination thereof. In still another aspect ofthis embodiment, a polynucleotide molecule encodes a PAR2 ligand domaincomprising a non-naturally occurring PAR2 ligand domain variant of SEQID NO: 24, such as, e.g., a conservative PAR2 ligand domain variant ofSEQ ID NO: 24, a non-conservative PAR2 ligand domain variant of SEQ IDNO: 24 or a PAR2 ligand domain peptidomimetic of SEQ ID NO: 24, or anycombination thereof. In other aspects of this embodiment, apolynucleotide molecule encodes a PAR2 ligand domain comprising SEQ IDNO: 24 or SEQ ID NO: 25.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR2 ligand domain comprising a polypeptide having, e.g., at least 50%amino acid identity with SEQ ID NO: 24, at least 67% amino acid identitywith the SEQ ID NO: 24, or at least 83% amino acid identity with SEQ IDNO: 24. In still other aspects of this embodiment, a polynucleotidemolecule encodes a PAR2 ligand domain comprising a polypeptide having,e.g., at most 50% amino acid identity with SEQ ID NO: 24, at most 67%amino acid identity with the SEQ ID NO: 24, at most 83% amino acididentity with SEQ ID NO: 24.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR2 ligand domain comprising a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid substitutions relative toSEQ ID NO: 24. In still other aspects of this embodiment, apolynucleotide molecule encodes a PAR2 ligand domain comprising apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to SEQ ID NO: 24. Inyet other aspects of this embodiment, a polynucleotide molecule encodesa PAR2 ligand domain comprising a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine or ten non-contiguousamino acid additions relative to SEQ ID NO: 24. In yet other aspects ofthis embodiment, a polynucleotide molecule encodes a PAR2 ligand domaincomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acid additionsrelative to SEQ ID NO: 24. In still other aspects of this embodiment, apolynucleotide molecule encodes a PAR2 ligand domain comprising apolypeptide having, e.g., at most one, two or three non-contiguous aminoacid deletions relative to SEQ ID NO: 24. In still other aspects of thisembodiment, a polynucleotide molecule encodes a PAR2 ligand domaincomprising a polypeptide having, e.g., at least one, two or threenon-contiguous amino acid deletions relative to SEQ ID NO: 24.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR2 ligand domain comprising a polypeptide having, e.g., at most two,three or four contiguous amino acid substitutions relative to SEQ ID NO:24. In still other aspects of this embodiment, a polynucleotide moleculeencodes a PAR2 ligand domain comprising a polypeptide having, e.g., atleast two, three or four contiguous amino acid substitutions relative toSEQ ID NO: 24. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a PAR2 ligand domain comprising a polypeptide having,e.g., at most two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 24. In yet otheraspects of this embodiment, a polynucleotide molecule encodes a PAR2ligand domain comprising a polypeptide having, e.g., at least two,three, four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 24. In still other aspects of thisembodiment, a polynucleotide molecule encodes a PAR2 ligand domaincomprising a polypeptide having, e.g., at most two or three contiguousamino acid deletions relative to SEQ ID NO: 24. In still other aspectsof this embodiment, a polynucleotide molecule encodes a PAR2 liganddomain comprising a polypeptide having, e.g., at least two or threecontiguous amino acid deletions relative to SEQ ID NO: 24.

In still another embodiment, a polynucleotide molecule encodes a PARligand domain comprising a PAR3 ligand domain. In an aspect of thisembodiment, a polynucleotide molecule encodes a PAR3 ligand domaincomprising SEQ ID NO: 26. In another aspect of this embodiment, apolynucleotide molecule encodes a PAR3 ligand domain comprising anaturally occurring PAR3 ligand domain variant, such as, e.g., a PAR3ligand domain isoform or a PAR3 ligand domain subtype. In another aspectof this embodiment, a polynucleotide molecule encodes a PAR3 liganddomain comprising a naturally occurring PAR3 ligand domain variant ofSEQ ID NO: 26, such as, e.g., a PAR3 ligand domain isoform of SEQ ID NO:26 or a PAR3 ligand domain subtype of SEQ ID NO: 26. In still anotheraspect of this embodiment, a polynucleotide molecule encodes a PAR3ligand domain comprising a non-naturally occurring PAR3 ligand domainvariant, such as, e.g., a conservative PAR3 ligand domain variant, anon-conservative PAR3 ligand domain variant or a PAR3 ligand domainpeptidomimetic, or any combination thereof. In still another aspect ofthis embodiment, a polynucleotide molecule encodes a PAR3 ligand domaincomprising a non-naturally occurring PAR3 ligand domain variant of SEQID NO: 26, such as, e.g., a conservative PAR3 ligand domain variant ofSEQ ID NO: 26, a non-conservative PAR3 ligand domain variant of SEQ IDNO: 26 or a PAR3 ligand domain peptidomimetic of SEQ ID NO: 26, or anycombination thereof. In other aspects of this embodiment, apolynucleotide molecule encodes a PAR3 ligand domain comprising SEQ IDNO: 26 or SEQ ID NO: 27.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR3 ligand domain comprising a polypeptide having, e.g., at least 50%amino acid identity with SEQ ID NO: 26, at least 67% amino acid identitywith the SEQ ID NO: 26, or at least 83% amino acid identity with SEQ IDNO: 26. In still other aspects of this embodiment, a polynucleotidemolecule encodes a PAR3 ligand domain comprising a polypeptide having,e.g., at most 50% amino acid identity with SEQ ID NO: 26, at most 67%amino acid identity with the SEQ ID NO: 26, at most 83% amino acididentity with SEQ ID NO: 26.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR3 ligand domain comprising a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid substitutions relative toSEQ ID NO: 26. In still other aspects of this embodiment, apolynucleotide molecule encodes a PAR3 ligand domain comprising apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to SEQ ID NO: 26. Inyet other aspects of this embodiment, a polynucleotide molecule encodesa PAR3 ligand domain comprising a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine or ten non-contiguousamino acid additions relative to SEQ ID NO: 26. In yet other aspects ofthis embodiment, a polynucleotide molecule encodes a PAR3 ligand domaincomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acid additionsrelative to SEQ ID NO: 26. In still other aspects of this embodiment, apolynucleotide molecule encodes a PAR3 ligand domain comprising apolypeptide having, e.g., at most one, two or three non-contiguous aminoacid deletions relative to SEQ ID NO: 26. In still other aspects of thisembodiment, a polynucleotide molecule encodes a PAR3 ligand domaincomprising a polypeptide having, e.g., at least one, two or threenon-contiguous amino acid deletions relative to SEQ ID NO: 26.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR3 ligand domain comprising a polypeptide having, e.g., at most two,three or four contiguous amino acid substitutions relative to SEQ ID NO:26. In still other aspects of this embodiment, a polynucleotide moleculeencodes a PAR3 ligand domain comprising a polypeptide having, e.g., atleast two, three or four contiguous amino acid substitutions relative toSEQ ID NO: 26. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a PAR3 ligand domain comprising a polypeptide having,e.g., at most two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 26. In yet otheraspects of this embodiment, a polynucleotide molecule encodes a PAR3ligand domain comprising a polypeptide having, e.g., at least two,three, four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 26. In still other aspects of thisembodiment, a polynucleotide molecule encodes a PAR3 ligand domaincomprising a polypeptide having, e.g., at most two or three contiguousamino acid deletions relative to SEQ ID NO: 26. In still other aspectsof this embodiment, a polynucleotide molecule encodes a PAR3 liganddomain comprising a polypeptide having, e.g., at least two or threecontiguous amino acid deletions relative to SEQ ID NO: 26.

In still another embodiment, a polynucleotide molecule encodes a PARligand domain comprising a PAR4 ligand domain. In an aspect of thisembodiment, a polynucleotide molecule encodes a PAR4 ligand domaincomprising SEQ ID NO: 28. In another aspect of this embodiment, apolynucleotide molecule encodes a PAR4 ligand domain comprising anaturally occurring PAR4 ligand domain variant, such as, e.g., a PAR4ligand domain isoform or a PAR4 ligand domain subtype. In another aspectof this embodiment, a polynucleotide molecule encodes a PAR4 liganddomain comprising a naturally occurring PAR4 ligand domain variant ofSEQ ID NO: 28, such as, e.g., a PAR4 ligand domain isoform of SEQ ID NO:28 or a PAR4 ligand domain subtype of SEQ ID NO: 28. In still anotheraspect of this embodiment, a polynucleotide molecule encodes a PAR4ligand domain comprising a non-naturally occurring PAR4 ligand domainvariant, such as, e.g., a conservative PAR4 ligand domain variant, anon-conservative PAR4 ligand domain variant or a PAR4 ligand domainpeptidomimetic, or any combination thereof. In still another aspect ofthis embodiment, a polynucleotide molecule encodes a PAR4 ligand domaincomprising a non-naturally occurring PAR4 ligand domain variant of SEQID NO: 28, such as, e.g., a conservative PAR4 ligand domain variant ofSEQ ID NO: 28, a non-conservative PAR4 ligand domain variant of SEQ IDNO: 28 or a PAR4 ligand domain peptidomimetic of SEQ ID NO: 28, or anycombination thereof. In other aspects of this embodiment, apolynucleotide molecule encodes a PAR4 ligand domain comprising SEQ IDNO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 or SEQ IDNO: 47.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR4 ligand domain comprising a polypeptide having, e.g., at least 50%amino acid identity with SEQ ID NO: 28, at least 67% amino acid identitywith the SEQ ID NO: 28, or at least 83% amino acid identity with SEQ IDNO: 28. In still other aspects of this embodiment, a polynucleotidemolecule encodes a PAR4 ligand domain comprising a polypeptide having,e.g., at most 50% amino acid identity with SEQ ID NO: 28, at most 67%amino acid identity with the SEQ ID NO: 28, at most 83% amino acididentity with SEQ ID NO: 28.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR4 ligand domain comprising a polypeptide having, e.g., at most one,two, three or four non-contiguous amino acid substitutions relative toSEQ ID NO: 28. In still other aspects of this embodiment, apolynucleotide molecule encodes a PAR4 ligand domain comprising apolypeptide having, e.g., at least one, two, three or fournon-contiguous amino acid substitutions relative to SEQ ID NO: 28. Inyet other aspects of this embodiment, a polynucleotide molecule encodesa PAR4 ligand domain comprising a polypeptide having, e.g., at most one,two, three, four, five, six, seven, eight, nine or ten non-contiguousamino acid additions relative to SEQ ID NO: 28. In yet other aspects ofthis embodiment, a polynucleotide molecule encodes a PAR4 ligand domaincomprising a polypeptide having, e.g., at least one, two, three, four,five, six, seven, eight, nine or ten non-contiguous amino acid additionsrelative to SEQ ID NO: 28. In still other aspects of this embodiment, apolynucleotide molecule encodes a PAR4 ligand domain comprising apolypeptide having, e.g., at most one, two or three non-contiguous aminoacid deletions relative to SEQ ID NO: 28. In still other aspects of thisembodiment, a polynucleotide molecule encodes a PAR4 ligand domaincomprising a polypeptide having, e.g., at least one, two or threenon-contiguous amino acid deletions relative to SEQ ID NO: 28.

In other aspects of this embodiment, a polynucleotide molecule encodes aPAR4 ligand domain comprising a polypeptide having, e.g., at most two,three or four contiguous amino acid substitutions relative to SEQ ID NO:28. In still other aspects of this embodiment, a polynucleotide moleculeencodes a PAR4 ligand domain comprising a polypeptide having, e.g., atleast two, three or four contiguous amino acid substitutions relative toSEQ ID NO: 28. In yet other aspects of this embodiment, a polynucleotidemolecule encodes a PAR4 ligand domain comprising a polypeptide having,e.g., at most two, three, four, five, six, seven, eight, nine or tencontiguous amino acid additions relative to SEQ ID NO: 28. In yet otheraspects of this embodiment, a polynucleotide molecule encodes a PAR4ligand domain comprising a polypeptide having, e.g., at least two,three, four, five, six, seven, eight, nine or ten contiguous amino acidadditions relative to SEQ ID NO: 28. In still other aspects of thisembodiment, a polynucleotide molecule encodes a PAR4 ligand domaincomprising a polypeptide having, e.g., at most two or three contiguousamino acid deletions relative to SEQ ID NO: 28. In still other aspectsof this embodiment, a polynucleotide molecule encodes a PAR4 liganddomain comprising a polypeptide having, e.g., at least two or threecontiguous amino acid deletions relative to SEQ ID NO: 28.

In yet another embodiment, a polynucleotide molecule encoding a modifiedClostridial toxin disclosed in the present specification can furthercomprise a polynucleotide molecule encoding a flexible region comprisinga flexible spacer. In another embodiment, a polynucleotide moleculeencoding a modified Clostridial toxin disclosed in the presentspecification can further comprise a polynucleotide molecule encoding aflexible region comprising a plurality of flexible spacers in tandem. Inaspects of this embodiment, a polynucleotide molecule encoding aflexible region can comprise in tandem, e.g., at least 1 G-spacer, atleast 2 G-spacers, at least 3 G-spacers, at least 4 G-spacers or atleast 5 G-spacers. In other aspects of this embodiment, a polynucleotidemolecule encoding a flexible region can comprise in tandem, e.g., atmost 1 G-spacer, at most 2 G-spacers, at most 3 G-spacers, at most 4G-spacers or at most 5 G-spacers. In still other aspects of thisembodiment, a polynucleotide molecule encoding a flexible region cancomprise in tandem, e.g., at least 1 A-spacer, at least 2 A-spacers, atleast 3 A-spacers, at least 4 A-spacers or at least 5 A-spacers. Instill other aspects of this embodiment, a polynucleotide moleculeencoding a flexible region can comprise in tandem, e.g., at most 1A-spacer, at most 2 A-spacers, at most 3 A-spacers, at most 4 A-spacersor at most 5 A-spacers. In another aspect of this embodiment, apolynucleotide molecule encoding a modified Clostridial toxin cancomprise a polynucleotide molecule encoding a flexible region comprisingone or more copies of the same flexible spacers, one or more copies ofdifferent flexible-spacers region, or any combination thereof.

In yet another embodiment, a polynucleotide molecule encoding a modifiedClostridial toxin disclosed in the present specification can furthercomprises a polynucleotide molecule encoding an epitope-binding region.In another embodiment, a polynucleotide molecule encoding a modifiedClostridial toxin disclosed in the present specification can furthercomprises a polynucleotide molecule encoding a plurality ofepitope-binding regions. In aspects of this embodiment, a polynucleotidemolecule encoding a modified Clostridial toxin can comprise, e.g., atleast 1 polynucleotide molecule encoding an epitope-binding region, atleast 2 polynucleotide molecules encoding epitope-binding regions, atleast 3 polynucleotide molecules encoding epitope-binding regions, atleast 4 polynucleotide molecules encoding epitope-binding regions or atleast 5 polynucleotide molecules encoding epitope-binding regions. Inother aspects of this embodiment, a polynucleotide molecule encoding amodified Clostridial toxin can comprise, e.g., at most 1 polynucleotidemolecule encoding an epitope-binding region, at most 2 polynucleotidemolecules encoding epitope-binding regions, at most 3 polynucleotidemolecules encoding epitope-binding regions, at most 4 polynucleotidemolecules encoding epitope-binding regions or at most 5 polynucleotidemolecules encoding epitope-binding regions. In another aspect of thisembodiment, a polynucleotide molecule encoding a modified Clostridialtoxin can comprise one or more copies of the same polynucleotidemolecules encoding epitope-binding region, one or more copies ofdifferent polynucleotide molecules encoding epitope-binding region, orany combination thereof. The location of a polynucleotide moleculeencoding an epitope-binding region can be in various positions,including, without limitation, at the amino terminus of a modifiedClostridial toxin, within a modified Clostridial toxin, or at thecarboxyl terminus of a modified Clostridial toxin.

In yet another embodiment, polynucleotide molecules encoding a modifiedClostridial toxin disclosed in the present specification can furthercomprise a polynucleotide molecule encoding an exogenous proteasecleavage site. In another embodiment, a polynucleotide molecule encodinga modified Clostridial toxin disclosed in the present specification canfurther comprises a plurality of polynucleotide molecules encodingexogenous protease cleavage sites. In aspects of this embodiment, apolynucleotide molecule encoding a modified Clostridial toxin cancomprise, e.g., at least 1 polynucleotide molecule encoding an exogenousprotease cleavage site, at least 2 polynucleotide molecules encodingexogenous protease cleavage sites, at least 3 polynucleotide moleculesencoding exogenous protease cleavage sites, at least 4 polynucleotidemolecules encoding exogenous protease cleavage sites or at least 5polynucleotide molecules encoding exogenous protease cleavage sites. Inother aspects of this embodiment, polynucleotide molecules encoding amodified Clostridial toxin can comprise, e.g., at most 1 polynucleotidemolecule encoding an exogenous protease cleavage site, at most 2polynucleotide molecules encoding exogenous protease cleavage sites, atmost 3 polynucleotide molecules encoding exogenous protease cleavagesites, at most 4 polynucleotide molecules encoding exogenous proteasecleavage sites or at most 5 polynucleotide molecules encoding exogenousprotease cleavage sites. In another aspect of this embodiment, apolynucleotide molecule encoding a modified Clostridial toxin cancomprise one or more copies of the same exogenous protease cleavagesite, one or more copies of different exogenous protease cleavage site,or any combination thereof.

In yet another embodiment, a polynucleotide molecule encoding anexogenous protease cleavage site is located between a polynucleotidemolecule encoding an epitope-binding peptide and a polynucleotidemolecule encoding a modified Clostridial toxin. In other aspects of thisembodiment, a polynucleotide molecule encoding a bovine enterokinasecleavage site is located between a polynucleotide molecule encoding anepitope-binding region and a polynucleotide molecule encoding a modifiedClostridial toxin, a polynucleotide molecule encoding a Tobacco EtchVirus protease cleavage site is located between a polynucleotidemolecule encoding an epitope-binding region and a polynucleotidemolecule encoding a modified Clostridial toxin, a polynucleotidemolecule encoding a Human Rhinovirus 3C protease cleavage site islocated between a polynucleotide molecule encoding an epitope-bindingregion and a polynucleotide molecule encoding a modified Clostridialtoxin, a polynucleotide molecule encoding a SUMO/ULP-1 protease cleavagesite is located between a polynucleotide molecule encoding anepitope-binding region and a polynucleotide molecule encoding a modifiedClostridial toxin, a polynucleotide molecule encoding a Thrombinprotease cleavage site is located between a polynucleotide moleculeencoding an epitope-binding region and a polynucleotide moleculeencoding a modified Clostridial toxin, or a polynucleotide moleculeencoding a Coagulation Factor Xa protease cleavage site is locatedbetween a polynucleotide molecule encoding an epitope-binding region anda polynucleotide molecule encoding a modified Clostridial toxin. Inother aspects of the embodiment, a polynucleotide molecule encoding thebovine enterokinase protease cleavage site of SEQ ID NO: 50 is locatedbetween a polynucleotide molecule encoding an epitope-binding region anda polynucleotide molecule encoding a modified Clostridial toxin. Inother aspects of the embodiment, a polynucleotide molecule encoding theTobacco Etch Virus protease cleavage site of SEQ ID NO: 51, SEQ ID NO:52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ IDNO: 57, SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60 is located betweena polynucleotide molecule encoding an epitope-binding region and apolynucleotide molecule encoding a modified Clostridial toxin. In stillother aspects of the embodiment, a polynucleotide molecule encoding theHuman Rhinovirus 3C protease cleavage site of SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66 islocated between a polynucleotide molecule encoding an epitope-bindingregion and a polynucleotide molecule encoding a modified Clostridialtoxin. In yet other aspects of the embodiment, a polynucleotide moleculeencoding the SUMO/ULP-1 protease cleavage site of SEQ ID NO: 67 islocated between a polynucleotide molecule encoding an epitope-bindingregion and a polynucleotide molecule encoding a modified Clostridialtoxin. In further other aspects of the embodiment, a polynucleotidemolecule encoding the Thrombin protease cleavage site of SEQ ID NO: 68,SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ IDNO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82 islocated between a polynucleotide molecule encoding an epitope-bindingregion and a polynucleotide molecule encoding a modified Clostridialtoxin. In other aspects of the embodiment, a polynucleotide moleculeencoding the Coagulation Factor Xa protease cleavage site of SEQ ID NO:83 or SEQ ID NO: 84 is located between a polynucleotide moleculeencoding an epitope-binding region and a polynucleotide moleculeencoding a modified Clostridial toxin.

In yet another embodiment, a polynucleotide molecule encoding anexogenous protease cleavage site is located within a polynucleotidemolecule encoding the di-chain loop of a modified Clostridial toxin. Inaspects of this embodiment, a polynucleotide molecule encoding a bovineenterokinase cleavage site is located within a polynucleotide moleculeencoding the di-chain loop of a modified Clostridial toxin, apolynucleotide molecule encoding a Tobacco Etch Virus protease cleavagesite is located within a polynucleotide molecule encoding the di-chainloop of a modified Clostridial toxin, a polynucleotide molecule encodinga Human Rhinovirus 3C protease cleavage site is located within apolynucleotide molecule encoding the di-chain loop of a modifiedClostridial toxin, a polynucleotide molecule encoding a SUMO/ULP-1protease cleavage site is located within a polynucleotide moleculeencoding the di-chain loop of a modified Clostridial toxin, apolynucleotide molecule encoding a Thrombin protease cleavage site islocated within a polynucleotide molecule encoding the di-chain loop of amodified Clostridial toxin, or a polynucleotide molecule encoding aCoagulation Factor Xa protease cleavage site is located within apolynucleotide molecule encoding the di-chain loop of a modifiedClostridial toxin. In other aspects of the embodiment, a polynucleotidemolecule encoding the bovine enterokinase protease cleavage site of SEQID NO: 50 is located within a polynucleotide molecule encoding thedi-chain loop of a modified Clostridial toxin. In other aspects of theembodiment, a polynucleotide molecule encoding the Tobacco Etch Virusprotease cleavage site of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO: 59 or SEQ ID NO: 60 is located within a polynucleotidemolecule encoding the di-chain loop of a modified Clostridial toxin. Instill other aspects of the embodiment, a polynucleotide moleculeencoding the Human Rhinovirus 3C protease cleavage site of SEQ ID NO:61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ IDNO: 66 is located within a polynucleotide molecule encoding the di-chainloop of a modified Clostridial toxin. In yet other aspects of theembodiment, a polynucleotide molecule encoding the SUMO/ULP-1 proteasecleavage site of SEQ ID NO: 67 is located within a polynucleotidemolecule encoding the di-chain loop of a modified Clostridial toxin. Infurther other aspects of the embodiment, a polynucleotide moleculeencoding the Thrombin protease cleavage site of SEQ ID NO: 68, SEQ IDNO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82 is locatedwithin a polynucleotide molecule encoding the di-chain loop of amodified Clostridial toxin. In other aspects of the embodiment, apolynucleotide molecule encoding the Coagulation Factor Xa proteasecleavage site of SEQ ID NO: 83 or SEQ ID NO: 84 is located within apolynucleotide molecule encoding the di-chain loop of a modifiedClostridial toxin.

Another aspect of the present invention provides a method of producing amodified Clostridial toxin comprising a PAR ligand domain; a Clostridialtoxin enzymatic domain; a Clostridial toxin translocation domain; and aClostridial toxin binding domain, such method comprising the step ofexpressing a polynucleotide molecule encoding a modified Clostridialtoxin in a cell. Another aspect of the present invention provides amethod of producing a modified Clostridial toxin comprising a PAR liganddomain; a Clostridial toxin enzymatic domain; a Clostridial toxintranslocation domain; and a Clostridial toxin binding domain, suchmethod comprising the steps of introducing an expression constructcomprising a polynucleotide molecule encoding a modified Clostridialtoxin into a cell and expressing the expression construct in the cell.

The methods disclosed in the present specification include, in part, aClostridial toxin. It is envisioned that any and all Clostridial toxinsdisclosed in the present specification can be produced using the methodsdisclosed in the present specification. Thus, aspects of this embodimentinclude producing, without limitation, naturally occurring Clostridialtoxins, naturally occurring Clostridial toxins variants, such as, e.g.,Clostridial toxins isoforms and Clostridial toxins subtypes,non-naturally occurring Clostridial toxins variants, such as, e.g.,conservative Clostridial toxins variants, non-conservative Clostridialtoxins variants and Clostridial toxins fragments thereof, or anycombination thereof.

The methods disclosed in the present specification include, in part, aPAR binding domain. It is envisioned that any and all PAR bindingdomains disclosed in the present specification can be produced using themethods disclosed in the present specification. Thus, aspects of thisembodiment include producing, without limitation, naturally occurringPAR binding domains, naturally occurring PAR binding domain variants,such as, e.g., PAR binding domain isoforms and PAR binding domainsubtypes, non-naturally occurring PAR binding domain variants, such as,e.g., conservative PAR binding domain variants, non-conservative PARbinding domain variants and PAR binding domain fragments thereof, or anycombination thereof.

The methods disclosed in the present specification include, in part, apolynucleotide molecule. It is envisioned that any and allpolynucleotide molecules disclosed in the present specification can beused. Thus, aspects of this embodiment include, without limitation,polynucleotide molecules encoding naturally occurring Clostridialtoxins; polynucleotide molecules encoding naturally occurringClostridial toxins variants, such as, e.g., Clostridial toxins isoformsand Clostridial toxins subtypes; polynucleotide molecules encodingnon-naturally occurring Clostridial toxins variants, such as, e.g.,conservative Clostridial toxins variants, non-conservative Clostridialtoxins variants and Clostridial toxins fragments thereof, or anycombination thereof.

The methods disclosed in the present specification include, in part, anexpression construct. An expression construct comprises a polynucleotidemolecule disclosed in the present specification operably-linked to anexpression vector useful for expressing the polynucleotide molecule in acell or cell-free extract. A wide variety of expression vectors can beemployed for expressing a polynucleotide molecule encoding a modifiedClostridial toxin, including, without limitation, a viral expressionvector; a prokaryotic expression vector; eukaryotic expression vectors,such as, e.g., a yeast expression vector, an insect expression vectorand a mammalian expression vector; and a cell-free extract expressionvector. It is further understood that expression vectors useful topractice aspects of these methods may include those which express amodified Clostridial toxin under control of a constitutive,tissue-specific, cell-specific or inducible promoter element, enhancerelement or both. Non-limiting examples of expression vectors, along withwell-established reagents and conditions for making and using anexpression construct from such expression vectors are readily availablefrom commercial vendors that include, without limitation, BDBiosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, SanDiego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMDBiosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; andStratagene, La Jolla, Calif. The selection, making and use of anappropriate expression vector are routine procedures well within thescope of one skilled in the art and from the teachings herein.

Thus, aspects of this embodiment include, without limitation, a viralexpression vector operably-linked to a polynucleotide molecule encodinga modified Clostridial toxin; a prokaryotic expression vectoroperably-linked to a polynucleotide molecule encoding a modifiedClostridial toxin; a yeast expression vector operably-linked to apolynucleotide molecule encoding a modified Clostridial toxin; an insectexpression vector operably-linked to a polynucleotide molecule encodinga modified Clostridial toxin; and a mammalian expression vectoroperably-linked to a polynucleotide molecule encoding a modifiedClostridial toxin. Other aspects of this embodiment include, withoutlimitation, expression constructs suitable for expressing a modifiedClostridial toxin disclosed in the present specification using acell-free extract comprising a cell-free extract expression vectoroperably linked to a polynucleotide molecule encoding a modifiedClostridial toxin. Other aspects of this embodiment include, withoutlimitation, expression constructs comprising polynucleotide moleculescomprising any one of SEQ ID NO: 109 through SEQ ID NO: 132 and SEQ IDNO: 136 through SEQ ID NO: 159. Other aspects of this embodimentinclude, without limitation, expression constructs comprisingpolynucleotide molecules encoding a modified Clostridial toxincomprising any one of SEQ ID NO: 85 through SEQ ID NO: 108.

The methods disclosed in the present specification include, in part, acell. It is envisioned that any and all cells can be used. Thus, aspectsof this embodiment include, without limitation, prokaryotic cellsincluding, without limitation, strains of aerobic, microaerophilic,capnophilic, facultative, anaerobic, gram-negative and gram-positivebacterial cells such as those derived from, e.g., Escherichia coli,Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis,Clostridia perfringens, Clostridia difficile, Caulobacter crescentus,Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls,Neisseria meningitidis, Pseudomonas fluorescens and Salmonellatyphimurium; and eukaryotic cells including, without limitation, yeaststrains, such as, e.g., those derived from Pichia pastoris, Pichiamethanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomycescerevisiae and Yarrowia lipolytica; insect cells and cell lines derivedfrom insects, such as, e.g., those derived from Spodoptera frugiperda,Trichoplusia ni, Drosophila melanogaster and Manduca sexta; andmammalian cells and cell lines derived from mammalian cells, such as,e.g., those derived from mouse, rat, hamster, porcine, bovine, equine,primate and human. Cell lines may be obtained from the American TypeCulture Collection (2004), at URL address www.atcc.org; EuropeanCollection of Cell Cultures (2204), at URL address www.ecacc.org.uk; andthe German Collection of Microorganisms and Cell Cultures (2004), at URLaddress www.dsmz.de. Non-limiting examples of specific protocols forselecting, making and using an appropriate cell line are described ine.g., INSECT CELL CULTURE ENGINEERING (Mattheus F. A. Goosen et al.eds., Marcel Dekker, 1993); INSECT CELL CULTURES: FUNDAMENTAL ANDAPPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer Academic Publishers,1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUES OF CELLCULTURE (Cambridge University Press, 1997); CELL AND TISSUE CULTURE:LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley and Sons,1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUE (Wiley-Liss, 4^(th) ed. 2000); ANIMAL CELL CULTURE: APRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press,3^(rd) ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001);BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press,2^(nd) ed. 2002); and CURRENT PROTOCOLS IN M OLECULAR BIOLOGY, supra,(2004). These protocols are routine procedures within the scope of oneskilled in the art and from the teaching herein.

The methods disclosed in the present specification include, in part,introducing into a cell a polynucleotide molecule. A polynucleotidemolecule introduced into a cell can be transiently or stably maintainedby that cell. Stably-maintained polynucleotide molecules may beextra-chromosomal and replicate autonomously, or they may be integratedinto the chromosomal material of the cell and replicatenon-autonomously. It is envisioned that any and all methods forintroducing a polynucleotide molecule disclosed in the presentspecification into a cell can be used. Methods useful for introducing anucleic acid molecule into a cell include, without limitation,chemical-mediated transfection such as, e.g., calciumphosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated,lipid-mediated, polyethyleneimine (PEI)-mediated, polylysine-mediatedand polybrene-mediated; physical-mediated transfection, such as, e.g.,biolistic particle delivery, microinjection, protoplast fusion andelectroporation; and viral-mediated transfection, such as, e.g.,retroviral-mediated transfection, see, e.g., Introducing Cloned Genesinto Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds.,Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed. 2001). Oneskilled in the art understands that selection of a specific method tointroduce an expression construct into a cell will depend, in part, onwhether the cell will transiently contain an expression construct orwhether the cell will stably contain an expression construct. Theseprotocols are routine procedures within the scope of one skilled in theart and from the teaching herein.

In an aspect of this embodiment, a chemical-mediated method, termedtransfection, is used to introduce a polynucleotide molecule encoding amodified Clostridial toxin into a cell. In chemical-mediated methods oftransfection the chemical reagent forms a complex with the nucleic acidthat facilitates its uptake into the cells. Such chemical reagentsinclude, without limitation, calcium phosphate-mediated, see, e.g.,Martin Jordan & Florian Worm, Transfection of adherent and suspendedcells by calcium phosphate, 33 (2) Methods 136-143 (2004);diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, cationicpolymer-mediated like polyethyleneimine (PEI)-mediated andpolylysine-mediated and polybrene-mediated, see, e.g., Chun Zhang etal., Polyethylenimine strategies for plasmid delivery to brain-derivedcells, 33 (2) Methods 144-150 (2004). Such chemical-mediated deliverysystems can be prepared by standard methods and are commerciallyavailable, see, e.g., CellPhect Transfection Kit (Amersham Biosciences,Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate andDEAE Dextran, (Stratagene, Inc., La Jolla, Calif.); Lipofectamine™Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen 500Transfection kit (Fermentas, Inc., Hanover, Md.), and SuperFect andEffectene Transfection Kits (Qiagen, Inc., Valencia, Calif.).

In another aspect of this embodiment, a physical-mediated method is usedto introduce a polynucleotide molecule encoding a modified Clostridialtoxin into a cell. Physical techniques include, without limitation,electroporation, biolistic and microinjection. Biolistics andmicroinjection techniques perforate the cell wall in order to introducethe nucleic acid molecule into the cell, see, e.g., Jeike E. Biewenga etal., Plasmid-mediated gene transfer in neurons using the biolisticstechnique, 71 (1) J. Neurosci. Methods. 67-75 (1997); and John O'Brien &Sarah C. R. Lummis, Biolistic and diolistic transfection: using the genegun to deliver DNA and lipophilic dyes into mammalian cells, 33 (2)Methods 121-125 (2004). Electroporation, also termedelectropermeabilization, uses brief, high-voltage, electrical pulses tocreate transient pores in the membrane through which the nucleic acidmolecules enter and can be used effectively for stable and transienttransfections of all cell types, see, e.g., M. Golzio et al., In vitroand in vivo electric field-mediated permeabilization, gene transfer, andexpression, 33 (2) Methods 126-135 (2004); and Oliver Gresch et al., Newnon-viral method for gene transfer into primary cells, 33 (2) Methods151-163 (2004).

In another aspect of this embodiment, a viral-mediated method, termedtransduction, is used to introduce a polynucleotide molecule encoding amodified Clostridial toxin into a cell. In viral-mediated methods oftransient transduction, the process by which viral particles infect andreplicate in a host cell has been manipulated in order to use thismechanism to introduce a nucleic acid molecule into the cell.Viral-mediated methods have been developed from a wide variety ofviruses including, without limitation, retroviruses, adenoviruses,adeno-associated viruses, herpes simplex viruses, picornaviruses,alphaviruses and baculoviruses, see, e.g., Armin Blesch, Lentiviral andMLV based retroviral vectors for ex vivo and in vivo gene transfer, 33(2) Methods 164-172 (2004); and Maurizio Federico, From lentiviruses tolentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M. Poeschla,Non-primate lentiviral vectors, 5 (5) Curr. Opin. Mol. Ther. 529-540(2003); Karim Benihoud et al, Adenovirus vectors for gene delivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H. Bueler, Adeno-associatedviral vectors for gene transfer and gene therapy, 380 (6) Biol. Chem.613-622 (1999); Chooi M. Lai et al., Adenovirus and adeno-associatedvirus vectors, 21 (12) DNA Cell Biol. 895-913 (2002); Edward A. Burtonet al., Gene delivery using herpes simplex virus vectors, 21 (12) DNACell Biol. 915-936 (2002); Paola Grandi et al., Targeting HSV ampliconvectors, 33 (2) Methods 179-186 (2004); Ilya Frolov et al.,Alphavirus-based expression vectors: strategies and applications, 93(21) Proc. Natl. Acad. Sci. U.S.A. 11371-11377 (1996); Markus U.Ehrengruber, Alphaviral gene transfer in neurobiology, 59 (1) Brain Res.Bull. 13-22 (2002); Thomas A. Kost & J. Patrick Condreay, Recombinantbaculoviruses as mammalian cell gene-delivery vectors, 20 (4) TrendsBiotechnol. 173-180 (2002); and A. Huser & C. Hofmann, Baculovirusvectors: novel mammalian cell gene-delivery vehicles and theirapplications, 3 (1) Am. J. Pharmacogenomics 53-63 (2003).

Adenoviruses, which are non-enveloped, double-stranded DNA viruses, areoften selected for mammalian cell transduction because adenoviruseshandle relatively large polynucleotide molecules of about 36 kb, areproduced at high titer, and can efficiently infect a wide variety ofboth dividing and non-dividing cells, see, e.g., Wim T. J. M. C. Hermenset al., Transient gene transfer to neurons and glia: analysis ofadenoviral vector performance in the CNS and PNS, 71 (1) J. Neurosci.Methods 85-98 (1997); and Hiroyuki Mizuguchi et al., Approaches forgenerating recombinant adenovirus vectors, 52 (3) Adv. Drug Deliv. Rev.165-176 (2001). Transduction using adenoviral-based system do notsupport prolonged protein expression because the nucleic acid moleculeis carried from an episome in the cell nucleus, rather than beingintegrated into the host cell chromosome. Adenoviral vector systems andspecific protocols for how to use such vectors are disclosed in, e.g.,ViraPower™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Adenoviral Expression System Instruction Manual25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and AdEasy™Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) andAdEasy™ Adenoviral Vector System Instruction Manual 064004f, Stratagene,Inc.

Nucleic acid molecule delivery can also use single-stranded RNAretroviruses, such as, e.g., oncoretroviruses and lentiviruses.Retroviral-mediated transduction often produce transduction efficienciesclose to 100%, can easily control the proviral copy number by varyingthe multiplicity of infection (MOI), and can be used to eithertransiently or stably transduce cells, see, e.g., Tiziana Tonini et al.,Transient production of retroviral- and lentiviral-based vectors for thetransduction of Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004);Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivoand in vivo gene transfer, 33 (2) Methods 164-172 (2004); FélixRecillas-Targa, Gene transfer and expression in mammalian cell lines andtransgenic animals, 267 Methods Mol. Biol. 417-433 (2004); and RolandWolkowicz et al., Lentiviral vectors for the delivery of DNA intomammalian cells, 246 Methods Mol. Biol. 391-411 (2004). Retroviralparticles consist of an RNA genome packaged in a protein capsid,surrounded by a lipid envelope. The retrovirus infects a host cell byinjecting its RNA into the cytoplasm along with the reversetranscriptase enzyme. The RNA template is then reverse transcribed intoa linear, double stranded cDNA that replicates itself by integratinginto the host cell genome. Viral particles are spread both vertically(from parent cell to daughter cells via the provirus) as well ashorizontally (from cell to cell via virions). This replication strategyenables long-term persistent expression since the nucleic acid moleculesof interest are stably integrated into a chromosome of the host cell,thereby enabling long-term expression of the protein. For instance,animal studies have shown that lentiviral vectors injected into avariety of tissues produced sustained protein expression for more than 1year, see, e.g., Luigi Naldini et al., In vivo gene delivery and stabletransduction of non-dividing cells by a lentiviral vector, 272 (5259)Science 263-267 (1996). The Oncoretroviruses-derived vector systems,such as, e.g., Moloney murine leukemia virus (MoMLV), are widely usedand infect many different non-dividing cells. Lentiviruses can alsoinfect many different cell types, including dividing and non-dividingcells and possess complex envelope proteins, which allows for highlyspecific cellular targeting.

Retroviral vectors and specific protocols for how to use such vectorsare disclosed in, e.g., U.S. patent Nos. Manfred Gossen & HermannBujard, Tight control of gene expression in eukaryotic cells bytetracycline-responsive promoters, U.S. Pat. No. 5,464,758 (Nov. 7,1995) and Hermann Bujard & Manfred Gossen, Methods for regulating geneexpression, U.S. Pat. No. 5,814,618 (Sep. 29, 1998) David S. Hogness,Polynucleotides encoding insect steroid hormone receptor polypeptidesand cells transformed with same, U.S. Pat. No. 5,514,578 (May 7, 1996)and David S. Hogness, Polynucleotide encoding insect ecdysone receptor,U.S. Pat. No. 6,245,531 (Jun. 12, 2001); Elisabetta Vegeto et al.,Progesterone receptor having C. terminal hormone binding domaintruncations, U.S. Pat. No. 5,364,791 (Nov. 15, 1994), Elisabetta Vegetoet al., Mutated steroid hormone receptors, methods for their use andmolecular switch for gene therapy, U.S. Pat. No. 5,874,534 (Feb. 23,1999) and Elisabetta Vegeto et al., Mutated steroid hormone receptors,methods for their use and molecular switch for gene therapy, U.S. Pat.No. 5,935,934 (Aug. 10, 1999). Furthermore, such viral delivery systemscan be prepared by standard methods and are commercially available, see,e.g., BD™ Tet-Off and Tet-On Gene Expression Systems (BDBiosciences-Clonetech, Palo Alto, Calif.) and BD™ Tet-Off and Tet-OnGene Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech,(Mar. 14, 2003), GeneSwitch™ System (Invitrogen, Inc., Carlsbad, Calif.)and GeneSwitch™ System A Mifepristone-Regulated Expression System forMammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002);ViraPower™ Lentiviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Lentiviral Expression System Instruction Manual25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and CompleteControl® Retroviral Inducible Mammalian Expression System (Stratagene,La Jolla, Calif.) and Complete Control® Retroviral Inducible MammalianExpression System Instruction Manual, 064005e.

The methods disclosed in the present specification include, in part,expressing a modified Clostridial toxin from a polynucleotide molecule.It is envisioned that any of a variety of expression systems may beuseful for expressing a modified Clostridial toxin from a polynucleotidemolecule disclosed in the present specification, including, withoutlimitation, cell-based systems and cell-free expression systems.Cell-based systems include, without limitation, viral expressionsystems, prokaryotic expression systems, yeast expression systems,baculoviral expression systems, insect expression systems and mammalianexpression systems. Cell-free systems include, without limitation, wheatgerm extracts, rabbit reticulocyte extracts and E. coli extracts andgenerally are equivalent to the method disclosed herein. Expression of apolynucleotide molecule using an expression system can include any of avariety of characteristics including, without limitation, inducibleexpression, non-inducible expression, constitutive expression,viral-mediated expression, stably-integrated expression, and transientexpression. Expression systems that include well-characterized vectors,reagents, conditions and cells are well-established and are readilyavailable from commercial vendors that include, without limitation,Ambion, Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.;BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science,Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limitingexamples on the selection and use of appropriate heterologous expressionsystems are described in e.g., PROTEIN EXPRESSION. A PRACTICALAPPROACH(S. J. Higgins and B. David Hames eds., Oxford University Press,1999); Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS.USING NATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and MeenaRai & Harish Padh, Expression Systems for Production of HeterologousProteins, 80 (9) CURRENT SCIENCE 1121-1128, (2001). These protocols areroutine procedures well within the scope of one skilled in the art andfrom the teaching herein.

A variety of cell-based expression procedures are useful for expressinga modified Clostridial toxin encoded by polynucleotide moleculedisclosed in the present specification. Examples included, withoutlimitation, viral expression systems, prokaryotic expression systems,yeast expression systems, baculoviral expression systems, insectexpression systems and mammalian expression systems. Viral expressionsystems include, without limitation, the ViraPower™ Lentiviral(Invitrogen, Inc., Carlsbad, Calif.), the Adenoviral Expression Systems(Invitrogen, Inc., Carlsbad, Calif.), the AdEasy™ XL Adenoviral VectorSystem (Stratagene, La Jolla, Calif.) and the ViraPort® Retroviral GeneExpression System (Stratagene, La Jolla, Calif.). Non-limiting examplesof prokaryotic expression systems include the Champion™ pET ExpressionSystem (EMD Biosciences-Novagen, Madison, Wis.), the TriEx™ BacterialExpression Systems (EMD Biosciences-Novagen, Madison, Wis.), theQIAexpress® Expression System (QIAGEN, Inc.), and the Affinity® ProteinExpression and Purification System (Stratagene, La Jolla, Calif.). Yeastexpression systems include, without limitation, the EasySelect™ PichiaExpression Kit (Invitrogen, Inc., Carlsbad, Calif.), the YES-Echo™Expression Vector Kits (Invitrogen, Inc., Carlsbad, Calif.) and theSpECTRA™ S. pombe Expression System (Invitrogen, Inc., Carlsbad,Calif.). Non-limiting examples of baculoviral expression systems includethe BaculoDirect™ (Invitrogen, Inc., Carlsbad, Calif.), the Bac-to-Bac®(Invitrogen, Inc., Carlsbad, Calif.), and the BD BaculoGold™ (BDBiosciences-Pharmigen, San Diego, Calif.). Insect expression systemsinclude, without limitation, the Drosophila Expression System (DES®)(Invitrogen, Inc., Carlsbad, Calif.), InsectSelect™ System (Invitrogen,Inc., Carlsbad, Calif.) and InsectDirect™ System (EMDBiosciences-Novagen, Madison, Wis.). Non-limiting examples of mammalianexpression systems include the T-REx™ (Tetracycline-RegulatedExpression) System (Invitrogen, Inc., Carlsbad, Calif.), the Flp-In™T-REx™ System (Invitrogen, Inc., Carlsbad, Calif.), the pcDNA™ system(Invitrogen, Inc., Carlsbad, Calif.), the pSecTag2 system (Invitrogen,Inc., Carlsbad, Calif.), the Exchanger® System, InterPlay™ Mammalian TAPSystem (Stratagene, La Jolla, Calif.), Complete Control® InducibleMammalian Expression System (Stratagene, La Jolla, Calif.) andLacSwitch® II Inducible Mammalian Expression System (Stratagene, LaJolla, Calif.).

Another procedure of expressing a modified Clostridial toxin encoded bypolynucleotide molecule disclosed in the present specification employs acell-free expression system such as, without limitation, prokaryoticextracts and eukaryotic extracts. Non-limiting examples of prokaryoticcell extracts include the RTS 100 E. coli HY Kit (Roche Applied Science,Indianapolis, Ind.), the ActivePro In Vitro Translation Kit (Ambion,Inc., Austin, Tex.), the EcoPro™ System (EMD Biosciences-Novagen,Madison, Wis.) and the Expressway™ Plus Expression System (Invitrogen,Inc., Carlsbad, Calif.). Eukaryotic cell extract include, withoutlimitation, the RTS 100 Wheat Germ CECF Kit (Roche Applied Science,Indianapolis, Ind.), the TnT® Coupled Wheat Germ Extract Systems(Promega Corp., Madison, Wis.), the Wheat Germ IVT™ Kit (Ambion, Inc.,Austin, Tex.), the Retic Lysate IVT™ Kit (Ambion, Inc., Austin, Tex.),the PROTEINscript® II System (Ambion, Inc., Austin, Tex.) and the TnT®Coupled Reticulocyte Lysate Systems (Promega Corp., Madison, Wis.).

Aspects of the present invention can also be described as follows:

-   1. A modified Clostridial toxin comprising:    -   a) a PAR ligand domain;    -   b) a Clostridial toxin enzymatic domain;    -   c) a Clostridial toxin translocation domain; and    -   d) a Clostridial toxin binding domain.-   2. The modified Clostridial toxin according to 1, wherein the PAR    ligand domain is operationally-linked to the amino terminus of the    Clostridial toxin enzymatic domain.-   3. The modified Clostridial toxin according to 2, wherein the    modified Clostridial toxin comprises an amino to carboxyl single    polypeptide linear order comprising the PAR ligand domain, the    Clostridial toxin enzymatic domain, the Clostridial toxin    translocation domain and the Clostridial toxin binding domain.-   4. The modified Clostridial toxin according to 2, wherein the    modified Clostridial toxin comprises an amino to carboxyl single    polypeptide linear order comprising the PAR ligand domain, the    Clostridial toxin enzymatic domain, the Clostridial toxin binding    domain and the Clostridial toxin translocation domain.-   5. The modified Clostridial toxin according to 1, wherein the PAR    ligand domain is operationally-linked to the amino terminus of the    Clostridial toxin translocation domain.-   6. The modified Clostridial toxin according to 5, wherein the    modified Clostridial toxin comprises an amino to carboxyl single    polypeptide linear order comprising the Clostridial toxin binding    domain, the Clostridial toxin enzymatic domain, the PAR ligand    domain and the Clostridial toxin translocation domain.-   7. The modified Clostridial toxin according to 5, wherein the    modified Clostridial toxin comprises an amino to carboxyl single    polypeptide linear order comprising the Clostridial toxin enzymatic    domain, the PAR ligand domain, the Clostridial toxin translocation    domain and the Clostridial toxin binding domain.-   8. The modified Clostridial toxin according to 1, wherein the PAR    ligand domain is operationally-linked to the amino terminus of the    Clostridial toxin binding domain.-   9. The modified Clostridial toxin according to 8, wherein the    modified Clostridial toxin comprises an amino to carboxyl single    polypeptide linear order comprising the Clostridial toxin enzymatic    domain, the PAR ligand domain, the Clostridial toxin binding domain    and the Clostridial toxin translocation domain.-   10. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin further comprises a protease cleavage    site; wherein cleavage of the protease cleavage site unmasks the PAR    ligand domain.-   11. The modified Clostridial toxin according to 1, wherein the PAR    ligand domain comprises a PAR1 ligand domain.-   12. The modified Clostridial toxin according to 11, wherein the PAR1    ligand domain comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,    SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID    NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO:    133.-   13. The modified Clostridial toxin according to 1, wherein the PAR    ligand domain comprises a PAR2 ligand domain.-   14. The modified Clostridial toxin according to 13, wherein the PAR2    ligand domain comprises SEQ ID NO: 24 or SEQ ID NO: 25.-   15. The modified Clostridial toxin according to 1, wherein the PAR    ligand domain comprises a PAR3 ligand domain.-   16. The modified Clostridial toxin according to 15, wherein the PAR3    ligand domain comprises SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO:    134.-   17. The modified Clostridial toxin according to 1, wherein the PAR    ligand domain comprises a PAR4 ligand domain.-   18. The modified Clostridial toxin according to 17, wherein the PAR4    ligand domain comprises SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30,    SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID    NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,    SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID    NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 135    or SEQ ID NO: 160.-   19. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin is a modified Botulinum toxin comprising    a PAR ligand domain, a Botulinum toxin enzymatic domain, a Botulinum    toxin translocation domain and a Botulinum toxin binding domain.-   20. The modified Clostridial toxin according to 19, wherein the    modified Botulinum toxin is a modified BoNT/A comprising a PAR    ligand domain, a BoNT/A enzymatic domain, a BoNT/A translocation    domain and a BoNT/A binding domain.-   21. The modified Clostridial toxin according to 19, wherein the    modified Botulinum toxin is a modified BoNT/B comprising a PAR    ligand domain, a BoNT/B enzymatic domain, a BoNT/B translocation    domain and a BoNT/B binding domain.-   22. The modified Clostridial toxin according to 19, wherein the    modified Botulinum toxin is a modified BoNT/C1 comprising a PAR    ligand domain, a BoNT/C1 enzymatic domain, a BoNT/C1 translocation    domain and a BoNT/C1 binding domain.-   23. The modified Clostridial toxin according to 19, wherein the    modified Botulinum toxin is a modified BoNT/D comprising a PAR    ligand domain, a BoNT/D enzymatic domain, a BoNT/D translocation    domain and a BoNT/D binding domain.-   24. The modified Clostridial toxin according to 19, wherein the    modified Botulinum toxin is a modified BoNT/E comprising a PAR    ligand domain, a BoNT/E enzymatic domain, a BoNT/E translocation    domain and a BoNT/E binding domain.-   25. The modified Clostridial toxin according to 19, wherein the    modified Botulinum toxin is a modified BoNT/F comprising a PAR    ligand domain, a BoNT/F enzymatic domain, a BoNT/F translocation    domain and a BoNT/F binding domain.-   26. The modified Clostridial toxin according to 19, wherein the    modified Botulinum toxin is a modified BoNT/G comprising a PAR    ligand domain, a BoNT/G enzymatic domain, a BoNT/G translocation    domain and a BoNT/G binding domain.-   27. The modified Clostridial toxin according to 1, wherein the    modified Clostridial toxin is a modified Tetanus toxin comprising a    PAR ligand domain, a Tetanus toxin enzymatic domain, a Tetanus toxin    translocation domain and a Tetanus toxin binding domain.-   28. A polynucleotide molecule encoding a modified Clostridial toxin,    the polynucleotide molecule comprising:    -   a) polynucleotide molecule encoding a PAR ligand domain;    -   b) polynucleotide molecule encoding a Clostridial toxin        enzymatic domain;    -   c) polynucleotide molecule encoding a Clostridial toxin        translocation domain; and    -   d) polynucleotide molecule encoding a Clostridial toxin binding        domain.-   29. The polynucleotide molecule according to 28, wherein the    polynucleotide molecule encodes a polypeptide comprising the PAR    ligand domain operationally-linked to the amino terminus of the    Clostridial toxin enzymatic domain.-   30. The polynucleotide molecule according to 29, wherein the    polynucleotide molecule encodes a modified Clostridial toxin    comprising an amino to carboxyl single polypeptide linear order    comprising the PAR ligand domain, the Clostridial toxin enzymatic    domain, the Clostridial toxin translocation domain and the    Clostridial toxin binding domain.-   31. The polynucleotide molecule according to 29, wherein the    polynucleotide molecule encodes a modified Clostridial toxin    comprising an amino to carboxyl single polypeptide linear order    comprising the PAR ligand domain, the Clostridial toxin enzymatic    domain, the Clostridial toxin binding domain and the Clostridial    toxin translocation domain.-   32. The polynucleotide molecule according to 28, wherein the    polynucleotide molecule encodes a polypeptide comprising the PAR    ligand domain operationally-linked to the amino terminus of the    Clostridial toxin translocation domain.-   33. The polynucleotide molecule according to 32, wherein the    polynucleotide molecule encodes a modified Clostridial toxin    comprising an amino to carboxyl single polypeptide linear order    comprising the Clostridial toxin binding domain, the Clostridial    toxin enzymatic domain, the PAR ligand domain and the Clostridial    toxin translocation domain.-   34. The polynucleotide molecule according to 32, wherein the    polynucleotide molecule encodes a modified Clostridial toxin    comprising an amino to carboxyl single polypeptide linear order    comprising the Clostridial toxin enzymatic domain, the PAR ligand    domain, the Clostridial toxin translocation domain and the    Clostridial toxin binding domain.-   35. The polynucleotide molecule according to 28, wherein the    polynucleotide molecule encodes a polypeptide comprising the PAR    ligand domain operationally-linked to the amino terminus of the    Clostridial toxin binding domain.-   36. The polynucleotide molecule according to 35, wherein the    polynucleotide molecule encodes a modified Clostridial toxin    comprising an amino to carboxyl single polypeptide linear order    comprising the Clostridial toxin enzymatic domain, the PAR ligand    domain, the Clostridial toxin binding domain and the Clostridial    toxin translocation domain.-   37. The polynucleotide molecule according to 28, wherein the    polynucleotide molecule further encodes a protease cleavage site;    wherein cleavage of the protease cleavage site unmasks the PAR    ligand domain.-   38. The polynucleotide molecule according to 28, wherein the    polynucleotide molecule encoding the PAR ligand domain comprises a    PAR1 ligand domain.-   39. The polynucleotide molecule according to 38, wherein the    polynucleotide molecule encodes the PAR1 ligand domain comprising    SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID    NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,    SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 133.-   40. The polynucleotide molecule according to 28, wherein the    polynucleotide molecule encoding the PAR ligand domain comprises a    PAR2 ligand domain.-   41. The polynucleotide molecule according to 40, wherein the    polynucleotide molecule encodes the PAR2 ligand domain comprises SEQ    ID NO: 24 or SEQ ID NO: 25.-   42. The polynucleotide molecule according to 28, wherein the    polynucleotide molecule encoding the PAR ligand domain comprises a    PAR3 ligand domain.-   43. The polynucleotide molecule according to 42, wherein the    polynucleotide molecule encodes the PAR3 ligand domain comprises SEQ    ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 134.-   44. The polynucleotide molecule according to 28, wherein the    polynucleotide molecule encoding the PAR ligand domain comprises a    PAR4 ligand domain.-   45. The polynucleotide molecule according to 44, wherein the    polynucleotide molecule encodes the PAR4 ligand domain comprises SEQ    ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:    32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ    ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:    41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ    ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 135 or SEQ ID NO: 160.-   46. The polynucleotide molecule according to 28, wherein the    polynucleotide molecule encoding the modified Clostridial toxin    comprises a polynucleotide molecule encoding a modified Botulinum    toxin comprising a PAR ligand domain, a Botulinum toxin enzymatic    domain, a Botulinum toxin translocation domain and a Botulinum toxin    binding domain.-   47. The modified Clostridial toxin according to 46, wherein the    polynucleotide molecule encoding the modified Botulinum toxin    comprises a polynucleotide molecule encoding a modified BoNT/A    comprising a PAR ligand domain, a BoNT/A enzymatic domain, a BoNT/A    translocation domain and a BoNT/A binding domain.-   48. The modified Clostridial toxin according to 46, wherein the    polynucleotide molecule encoding the modified Botulinum toxin    comprises a polynucleotide molecule encoding a modified BoNT/B    comprising a PAR ligand domain, a BoNT/B enzymatic domain, a BoNT/B    translocation domain and a BoNT/B binding domain.-   49. The modified Clostridial toxin according to 46, wherein the    polynucleotide molecule encoding the modified Botulinum toxin    comprises a polynucleotide molecule encoding a modified BoNT/C1    comprising a PAR ligand domain, a BoNT/C1 enzymatic domain, a    BoNT/C1 translocation domain and a BoNT/C1 binding domain.-   50. The modified Clostridial toxin according to 46, wherein the    polynucleotide molecule encoding the modified Botulinum toxin    comprises a polynucleotide molecule encoding a modified BoNT/D    comprising a PAR ligand domain, a BoNT/D enzymatic domain, a BoNT/D    translocation domain and a BoNT/D binding domain.-   51. The modified Clostridial toxin according to 46, wherein the    polynucleotide molecule encoding the modified Botulinum toxin    comprises a polynucleotide molecule encoding a modified BoNT/E    comprising a PAR ligand domain, a BoNT/E enzymatic domain, a BoNT/E    translocation domain and a BoNT/E binding domain.-   52. The modified Clostridial toxin according to 46, wherein the    polynucleotide molecule encoding the modified Botulinum toxin    comprises a polynucleotide molecule encoding a modified BoNT/F    comprising a PAR ligand domain, a BoNT/F enzymatic domain, a BoNT/F    translocation domain and a BoNT/F binding domain.-   53. The modified Clostridial toxin according to 46, wherein the    polynucleotide molecule encoding the modified Botulinum toxin    comprises a polynucleotide molecule encoding a modified BoNT/G    comprising a PAR ligand domain, a BoNT/G enzymatic domain, a BoNT/G    translocation domain and a BoNT/G binding domain.-   54. The modified Clostridial toxin according to 28, wherein the    polynucleotide molecule encoding the modified Clostridial toxin    comprises a polynucleotide molecule encoding a modified Tetanus    toxin comprising a PAR ligand domain, a Tetanus toxin enzymatic    domain, a Tetanus toxin translocation domain and a Tetanus toxin    binding domain.-   55. A method of producing a modified Clostridial toxin comprising    the step of expressing a modified Clostridial toxin encoded by a    polynucleotide molecule in a cell, wherein the modified Clostridial    toxin comprising a PAR ligand domain; a Clostridial toxin enzymatic    domain; a Clostridial toxin translocation domain; and a Clostridial    toxin binding domain.-   56. A methods of producing a modified Clostridial toxin comprising    the steps of:    -   a. introducing into a cell a polynucleotide molecule encoding a        modified Clostridial toxin comprising a PAR ligand domain; a        Clostridial toxin enzymatic domain; a Clostridial toxin        translocation domain; and a Clostridial toxin binding domain;        and    -   b. expressing the modified Clostridial toxin encoded by the        polynucleotide molecule.-   57. A modified Clostridial toxin comprising:    -   a) a PAR ligand domain;    -   b) a Clostridial toxin enzymatic domain;    -   c) a Clostridial toxin translocation domain; and    -   d) a non-Clostridial toxin binding domain.-   58. The modified Clostridial toxin according to 57, wherein the    non-Clostridial toxin binding domain is selected from the group    consisting of a Nerve growth factor (NGF), a Leukemia inhibitory    factor (LIF), a Basic fibroblast growth factor (bFGF), a    Brain-derived neurotrophic factor (BDNF), a Neurotrophin-3 (NT-3), a    Hydra head activator peptide (HHAP), a Transforming growth factor 1    (TGF-1), a Transforming growth factor 2 (TGF-2), a Transforming    growth factor 3(TGF-3), an Epidermal growth factor (EGF) or a    Ciliary neurotrophic factor (CNTF).-   59. The modified Clostridial toxin according to 57, wherein the    non-Clostridial toxin binding domain is selected from the group    consisting of a Tumor necrosis factor (TNF-), an Interleukin-1    (IL-1), an Interleukin-1 (IL-1) or an Interleukin-8 (IL-8).-   60. The modified Clostridial toxin according to 57, wherein the    non-Clostridial toxin binding domain is selected from the group    consisting of a Bradykinin, a Dynorphin, a β-endorphin, an    Etorphine, an Endomorphin-1, an Endomorphin-2, a Leu-enkephalin, a    Met-enkephalin, a Galanin, a Lofentanil or a Nociceptin.-   61. The modified Clostridial toxin according to 57, wherein the    non-Clostridial toxin binding domain is selected from the group    consisting of an antibody against the lactoseries carbohydrate    epitopes found on the surface of dorsal root ganglion neurons (e.g.    monoclonal antibodies 1B2 and LA4), an antibody against any of the    receptors for the binding domains given above or an antibody against    the surface expressed antigen Thyl (e.g. monoclonal antibody MRC    OX7).

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofdisclosed embodiments and are in no way intended to limit any of theembodiments disclosed in the present specification.

Example 1 Construction of BoNT/A-ED-PAR1Tb

This example illustrates how to make a modified Clostridial toxincomprising a PAR binding domain located at the amino terminus of thelight chain comprising the enzymatic domain.

A polynucleotide molecule (SEQ ID NO: 109) based on BoNT/A-ED-PAR1Tb(SEQ ID NO: 85) is synthesized using standard procedures (BlueHeron®Biotechnology, Bothell, Wash.). Oligonucleotides of 20 to 50 bases inlength are synthesized using standard phosphoramidite synthesis. Theseoligonucleotides are hybridized into double stranded duplexes that areligated together to assemble the full-length polynucleotide molecule.This polynucleotide molecule is cloned using standard molecular biologymethods into a pUCBHB1 vector at the SmaI site to generatepUCBHB1/BoNT/A-ED-PAR1Tb. The synthesized polynucleotide molecule isverified by sequencing using Big Dye Terminator™ Chemistry 3.1 (AppliedBiosystems, Foster City, Calif.) and an ABI 3100 sequencer (AppliedBiosystems, Foster City, Calif.).

If desired, an expression optimized polynucleotide molecule (SEQ ID NO:136) based on BoNT/A-ED-PAR1Tb (SEQ ID NO: 85) can be synthesized inorder to improve expression in an Escherichia coli strain. Thepolynucleotide molecule encoding the BoNT/A-ED-PAR1Tb can be modifiedto 1) contain synonymous codons typically present in nativepolynucleotide molecules of an Escherichia coli strain; 2) contain a G+Ccontent that more closely matches the average G+C content of nativepolynucleotide molecules found in an Escherichia coli strain; 3) reducepolymononucleotide regions found within the polynucleotide molecule;and/or 4) eliminate internal regulatory or structural sites found withinthe polynucleotide molecule, see, e.g., Lance E. Steward et al.Optimizing Expression of Active Botulinum Toxin Type E, PCT PatentSerial No. 2005/020578 (Jun. 9, 2005); Lance E. Steward et al.Optimizing Expression of Active Botulinum Toxin Type A, PCT PatentSerial No. 2005/027917 (Aug. 3, 2005). Once sequence optimization iscomplete, oligonucleotides of 20 to 50 bases in length are synthesizedusing standard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate pUCBHB1/BoNT/A-ED-PAR1Tb.The synthesized polynucleotide molecule is verified by sequencing usingBig Dye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City,Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,Calif.). Is so desired, optimization to a different organism, such as,e.g., a yeast strain, an insect cell-line or a mammalian cell line, canbe done, see, e.g., Steward, supra, PCT Patent Serial No. 2005/020578(Jun. 9, 2005); and Steward, supra, PCT Patent Serial No. 2005/027917(Aug. 3, 2005).

A similar cloning strategy is used to make pUCBHB1 cloning constructscomprising the polynucleotide molecule of SEQ ID NO: 110 or SEQ ID NO:137 encoding BoNT/A-ED-PAR1Xa of SEQ ID NO: 86; the polynucleotidemolecule of SEQ ID NO: 111 or SEQ ID NO: 138 encoding BoNT/A-ED-PAR2Tpof SEQ ID NO: 87; the polynucleotide molecule of SEQ ID NO: 112 or SEQID NO: 139 encoding BoNT/A-ED-PAR2Xa of SEQ ID NO: 88; thepolynucleotide molecule of SEQ ID NO: 113 or SEQ ID NO: 140 encodingBoNT/A-ED-PAR3Tb of SEQ ID NO: 89; the polynucleotide molecule of SEQ IDNO: 114 or SEQ ID NO: 141 encoding BoNT/A-ED-PAR3Xa of SEQ ID NO: 90;the polynucleotide molecule of SEQ ID NO: 115 or SEQ ID NO: 142 encodingBoNT/A-ED-PAR4Tb of SEQ ID NO: 91; and the polynucleotide molecule ofSEQ ID NO: 116 or SEQ ID NO: 143 encoding BoNT/A-ED-PAR4Xa of SEQ ID NO:92. In addition, one skilled in the art can modify Clostridial toxins,such as, e.g., BoNT/B, BoNT/C1, BoNT/D. BoNT/E, BoNT/F, BoNT/G and TeNT,so that these toxins possess the PAR attributes of the modified BoNT/Adescribed above and make them using similar cloning strategy.

To construct pET29/BoNT/A-ED-PAR1Tb, a pUCBHB1/BoNT/A-ED-PAR1Tbconstruct is digested with restriction endonucleases that 1) excise theinsert comprising the open reading frame of SEQ ID NO: 136 encodingBoNT/A-ED-PAR1Tb; and 2) enable this insert to be operably-linked to apET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert issubcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/BoNT/A-ED-PAR1Tb. The ligation mixture is transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 50 μg/mL of Kanamycin, and placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs are identified as Kanamycin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pET29 expression construct comprising thepolynucleotide molecule of SEQ ID NO: 136 encoding the BoNT/A-ED-PAR1Tbof SEQ ID NO: 85 operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide (FIG. 7).

A similar cloning strategy is used to make pET29 expression constructscomprising the polynucleotide molecule of SEQ ID NO: 109 encodingBoNT/A-ED-PAR1Tb of SEQ ID NO: 85; SEQ ID NO: 110 or SEQ ID NO: 137encoding BoNT/A-ED-PAR1Xa of SEQ ID NO: 86; the polynucleotide moleculeof SEQ ID NO: 111 or SEQ ID NO: 138 encoding BoNT/A-ED-PAR2Tp of SEQ IDNO: 87; the polynucleotide molecule of SEQ ID NO: 112 or SEQ ID NO: 139encoding BoNT/A-ED-PAR2Xa of SEQ ID NO: 88; the polynucleotide moleculeof SEQ ID NO: 113 or SEQ ID NO: 140 encoding BoNT/A-ED-PAR3Tb of SEQ IDNO: 89; the polynucleotide molecule of SEQ ID NO: 114 or SEQ ID NO: 141encoding BoNT/A-ED-PAR3Xa of SEQ ID NO: 90; the polynucleotide moleculeof SEQ ID NO: 115 or SEQ ID NO: 142 encoding BoNT/A-ED-PAR4Tb of SEQ IDNO: 91; and the polynucleotide molecule of SEQ ID NO: 116 or SEQ ID NO:143 encoding BoNT/A-ED-PAR4Xa of SEQ ID NO: 92.

Example 2 Construction of BoNT/A-TD-PAR1Tb

This example illustrates how to make a modified Clostridial toxincomprising a PAR binding domain located at the amino terminus of theheavy chain region comprising the translocation domain.

A polynucleotide molecule (SEQ ID NO: 117) based on BoNT/A-TD-PAR1Tb(SEQ ID NO: 93) is synthesized using standard procedures (BlueHeron®Biotechnology, Bothell, Wash.). Oligonucleotides of 20 to 50 bases inlength are synthesized using standard phosphoramidite synthesis. Theseoligonucleotides are hybridized into double stranded duplexes that areligated together to assemble the full-length polynucleotide molecule.This polynucleotide molecule is cloned using standard molecular biologymethods into a pUCBHB1 vector at the SmaI site to generatepUCBHB1/BoNT/A-TD-PAR1Tb. The synthesized polynucleotide molecule isverified by sequencing using Big Dye Terminator™ Chemistry 3.1 (AppliedBiosystems, Foster City, Calif.) and an ABI 3100 sequencer (AppliedBiosystems, Foster City, Calif.).

If desired, an expression optimized polynucleotide molecule (SEQ ID NO:144) based on BoNT/A-TD-PAR1Tb (SEQ ID NO: 93) can be synthesized inorder to improve expression in an Escherichia coli strain. The openreading frame comprising the polynucleotide molecule is optimized toimprove expression in an Escherichia coli strain. The polynucleotidemolecule encoding the BoNT/A-TD-PAR1Tb can be modified to 1) containsynonymous codons typically present in native polynucleotide moleculesof an Escherichia coli strain; 2) contain a G+C content that moreclosely matches the average G+C content of native polynucleotidemolecules found in an Escherichia coli strain; 3) reducepolymononucleotide regions found within the polynucleotide molecule;and/or 4) eliminate internal regulatory or structural sites found withinthe polynucleotide molecule, see, e.g., Lance E. Steward et al.Optimizing Expression of Active Botulinum Toxin Type E, PCT PatentSerial No. 2005/020578 (Jun. 9, 2005); Lance E. Steward et al.Optimizing Expression of Active Botulinum Toxin Type A, PCT PatentSerial No. 2005/027917 (Aug. 3, 2005). Once sequence optimization iscomplete, oligonucleotides of 20 to 50 bases in length are synthesizedusing standard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate BoNT/A-TD-PAR1Tb. Thesynthesized polynucleotide molecule is verified by sequencing using BigDye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.)and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.). Isso desired, optimization of the polynucleotide molecule encoding aBoNT/A-TD-PAR1Tb need not be performed, or optimization to a differentorganism, such as, e.g., a yeast strain, an insect cell-line or amammalian cell line, can be done instead, see, e.g., Steward, supra, PCTPatent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCTPatent Serial No. 2005/027917 (Aug. 3, 2005).

A similar cloning strategy is used to make pUCBHB1 cloning constructscomprising the polynucleotide molecule of SEQ ID NO: 118 or SEQ ID NO:145 encoding BoNT/A-TD-PAR1Xa of SEQ ID NO: 94; the polynucleotidemolecule of SEQ ID NO: 119 or SEQ ID NO: 146 encoding BoNT/A-TD-PAR2Tpof SEQ ID NO: 95; the polynucleotide molecule of SEQ ID NO: 120 or SEQID NO: 147 encoding BoNT/A-TD-PAR2Xa of SEQ ID NO: 96; thepolynucleotide molecule of SEQ ID NO: 121 or SEQ ID NO: 148 encodingBoNT/A-TD-PAR1Tb of SEQ ID NO: 97; the polynucleotide molecule of SEQ IDNO: 122 or SEQ ID NO: 149 encoding BoNT/A-TD-PAR3Xa of SEQ ID NO: 98;the polynucleotide molecule of SEQ ID NO: 123 or SEQ ID NO: 150 encodingBoNT/A-TD-PAR4Tb of SEQ ID NO: 99; and the polynucleotide molecule ofSEQ ID NO: 124 or SEQ ID NO: 151 encoding BoNT/A-TD-PAR4Xa of SEQ ID NO:100. In addition, one skilled in the art can modify Clostridial toxins,such as, e.g., BoNT/B, BoNT/C1, BoNT/D. BoNT/E, BoNT/F, BoNT/G and TeNT,so that these toxins possess the PAR attributes of the modified BoNT/Adescribed above and make them using similar cloning strategy.

To construct pET29/BoNT/A-TD-PAR1Tb, a pUCBHB1/BoNT/A-TD-PAR1Tbconstruct is digested with restriction endonucleases that 1) excise theinsert comprising the open reading frame of SEQ ID NO: 144 encodingBoNT/A-TD-PAR1Tb; and 2) enable this insert to be operably-linked to apET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert issubcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/BoNT/A-TD-PAR1Tb. The ligation mixture is transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 50 μg/mL of Kanamycin, and placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs are identified as Kanamycin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pET29 expression construct comprising thepolynucleotide molecule of SEQ ID NO: 144 encoding the BoNT/A-TD-PAR1Tbof SEQ ID NO: 93 operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide (FIG. 8).

A similar cloning strategy is used to make pET29 expression constructscomprising the polynucleotide molecule of SEQ ID NO: 117 encodingBoNT/A-TD-PAR1Xa of SEQ ID NO: 93; SEQ ID NO: 118 or SEQ ID NO: 145encoding BoNT/A-TD-PAR1Xa of SEQ ID NO: 94; the polynucleotide moleculeof SEQ ID NO: 119 or SEQ ID NO: 146 encoding BoNT/A-TD-PAR2Tp of SEQ IDNO: 95; the polynucleotide molecule of SEQ ID NO: 120 or SEQ ID NO: 147encoding BoNT/A-TD-PAR2Xa of SEQ ID NO: 96; the polynucleotide moleculeof SEQ ID NO: 121 or SEQ ID NO: 148 encoding BoNT/A-TD-PAR1Tb of SEQ IDNO: 97; the polynucleotide molecule of SEQ ID NO: 122 or SEQ ID NO: 149encoding BoNT/A-TD-PAR3Xa of SEQ ID NO: 98; the polynucleotide moleculeof SEQ ID NO: 123 or SEQ ID NO: 150 encoding BoNT/A-TD-PAR4Tb of SEQ IDNO: 99; and the polynucleotide molecule of SEQ ID NO: 124 or SEQ ID NO:151 encoding BoNT/A-TD-PAR4Xa of SEQ ID NO: 100.

Example 3 Construction of BoNT/A-BD-PAR1Tb

This example illustrates how to make a modified Clostridial toxincomprising a PAR binding domain located at the amino terminus of theheavy chain region comprising the binding domain.

A polynucleotide molecule (SEQ ID NO: 125) based on BoNT/A-BD-PAR1Tb(SEQ ID NO: 101) is synthesized using standard procedures (BlueHeron®Biotechnology, Bothell, Wash.). Oligonucleotides of 20 to 50 bases inlength are synthesized using standard phosphoramidite synthesis. Theseoligonucleotides are hybridized into double stranded duplexes that areligated together to assemble the full-length polynucleotide molecule.This polynucleotide molecule is cloned using standard molecular biologymethods into a pUCBHB1 vector at the SmaI site to generatepUCBHB1/BoNT/A-BD-PAR1Tb. The synthesized polynucleotide molecule isverified by sequencing using Big Dye Terminator™ Chemistry 3.1 (AppliedBiosystems, Foster City, Calif.) and an ABI 3100 sequencer (AppliedBiosystems, Foster City, Calif.).

If desired, an expression optimized polynucleotide molecule (SEQ ID NO:152) based on BoNT/A-BD-PAR1Tb (SEQ ID NO: 101) can be synthesized inorder to improve expression in an Escherichia coli strain. The openreading frame comprising the polynucleotide molecule is optimized toimprove expression in an Escherichia coli strain. The polynucleotidemolecule encoding the BoNT/A-BD-PAR1Tb can be modified to 1) containsynonymous codons typically present in native polynucleotide moleculesof an Escherichia coli strain; 2) contain a G+C content that moreclosely matches the average G+C content of native polynucleotidemolecules found in an Escherichia coli strain; 3) reducepolymononucleotide regions found within the polynucleotide molecule;and/or 4) eliminate internal regulatory or structural sites found withinthe polynucleotide molecule, see, e.g., Lance E. Steward et al.Optimizing Expression of Active Botulinum Toxin Type E, PCT PatentSerial No. 2005/020578 (Jun. 9, 2005); Lance E. Steward et al.Optimizing Expression of Active Botulinum Toxin Type A, PCT PatentSerial No. 2005/027917 (Aug. 3, 2005). Once sequence optimization iscomplete, oligonucleotides of 20 to 50 bases in length are synthesizedusing standard phosphoramidite synthesis. These oligonucleotides arehybridized into double stranded duplexes that are ligated together toassemble the full-length polynucleotide molecule. This polynucleotidemolecule is cloned using standard molecular biology methods into apUCBHB1 vector at the SmaI site to generate BoNT/A-BD-PAR1Tb. Thesynthesized polynucleotide molecule is verified by sequencing using BigDye Terminator™ Chemistry 3.1 (Applied Biosystems, Foster City, Calif.)and an ABI 3100 sequencer (Applied Biosystems, Foster City, Calif.). Isso desired, optimization of the polynucleotide molecule encoding aBoNT/A-BD-PAR1Tb need not be performed, or optimization to a differentorganism, such as, e.g., a yeast strain, an insect cell-line or amammalian cell line, can be done instead, see, e.g., Steward, supra, PCTPatent Serial No. 2005/020578 (Jun. 9, 2005); and Steward, supra, PCTPatent Serial No. 2005/027917 (Aug. 3, 2005).

A similar cloning strategy is used to make pUCBHB1 cloning constructscomprising the polynucleotide molecule of SEQ ID NO: 126 or SEQ ID NO:153 encoding BoNT/A-BD-PAR1Xa of SEQ ID NO: 102; the polynucleotidemolecule of SEQ ID NO: 127 or SEQ ID NO: 154 encoding BoNT/A-BD-PAR2Tpof SEQ ID NO: 103; the polynucleotide molecule of SEQ ID NO: 128 or SEQID NO: 155 encoding BoNT/A-BD-PAR2Xa of SEQ ID NO: 104; thepolynucleotide molecule of SEQ ID NO: 129 or SEQ ID NO: 156 encodingBoNT/A-BD-PAR3Tb of SEQ ID NO: 105; the polynucleotide molecule of SEQID NO: 130 or SEQ ID NO: 157 encoding BoNT/A-BD-PAR3Xa of SEQ ID NO:106; the polynucleotide molecule of SEQ ID NO: 131 or SEQ ID NO: 158encoding BoNT/A-BD-PAR4Tb of SEQ ID NO: 107; and the polynucleotidemolecule of SEQ ID NO: 132 or SEQ ID NO: 159 encoding BoNT/A-BD-PAR4Xaof SEQ ID NO: 108. In addition, one skilled in the art can modifyClostridial toxins, such as, e.g., BoNT/B, BoNT/C1, BoNT/D. BoNT/E,BoNT/F, BoNT/G and TeNT, so that these toxins possess the PAR attributesof the modified BoNT/A described above and make them using similarcloning strategy.

To construct pET29/BoNT/A-BD-PAR1Tb, a pUCBHB1/BoNT/A-BD-PAR1Tbconstruct is digested with restriction endonucleases that 1) excise theinsert comprising the open reading frame of SEQ ID NO: 152 encodingBoNT/A-BD-PAR1Tb; and 2) enable this insert to be operably-linked to apET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert issubcloned using a T4 DNA ligase procedure into a pET29 vector that isdigested with appropriate restriction endonucleases to yieldpET29/BoNT/A-BD-PAR1Tb. The ligation mixture is transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 50 μg/mL of Kanamycin, and placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs are identified as Kanamycin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pET29 expression construct comprising thepolynucleotide molecule of SEQ ID NO: 152 encoding the BoNT/A-BD-PAR1Tbof SEQ ID NO: 101 operably-linked to a carboxyl terminal polyhistidineaffinity binding peptide (FIG. 9).

A similar cloning strategy is used to make pET29 expression constructscomprising the polynucleotide molecule of SEQ ID NO: 125 encodingBoNT/A-BD-PAR1Tb of SEQ ID NO: 101; the polynucleotide molecule of SEQID NO: 126 or SEQ ID NO: 153 encoding BoNT/A-BD-PAR1Xa of SEQ ID NO:102; the polynucleotide molecule of SEQ ID NO: 127 or SEQ ID NO: 154encoding BoNT/A-BD-PAR2Tp of SEQ ID NO: 103; the polynucleotide moleculeof SEQ ID NO: 128 or SEQ ID NO: 155 encoding BoNT/A-BD-PAR2Xa of SEQ IDNO: 104; the polynucleotide molecule of SEQ ID NO: 129 or SEQ ID NO: 156encoding BoNT/A-BD-PAR3Tb of SEQ ID NO: 105; the polynucleotide moleculeof SEQ ID NO: 130 or SEQ ID NO: 157 encoding BoNT/A-BD-PAR3Xa of SEQ IDNO: 106; the polynucleotide molecule of SEQ ID NO: 131 or SEQ ID NO: 158encoding BoNT/A-BD-PAR4Tb of SEQ ID NO: 107; and the polynucleotidemolecule of SEQ ID NO: 132 or SEQ ID NO: 159 encoding BoNT/A-BD-PAR4Xaof SEQ ID NO: 108.

Example 4 Expression of Modified Clostridial Toxins in a Bacterial Cell

The following example illustrates a procedure useful for expressing anyof the modified Clostridial toxins disclosed in the presentspecification in a bacterial cell.

An expression construct, such as, e.g., pET29/BoNT/A-ED-PAR1Tb,pET29/BoNT/A-TD-PAR1Tb or pET29/BoNT/A-BD-PAR1Tb, see, e.g., Examples 1,2 and 3, is introduced into chemically competent E. coli BL21 (DE3)cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat-shocktransformation protocol. The heat-shock reaction is plated onto 1.5%Luria-Bertani agar plates (pH 7.0) containing 50 μg/mL of Kanamycin andis placed in a 37° C. incubator for overnight growth.Kanamycin-resistant colonies of transformed E. coli containing theexpression construct, such as, e.g., pET29/BoNT/A-ED-PAR1Tb,pET29/BoNT/A-TD-PAR1Tb or pET29/BoNT/A-BD-PAR1Tb, are used to inoculatea baffled flask containing 3.0 mL of PA-0.5G media containing 50 μg/mLof Kanamycin which is then placed in a 37° C. incubator, shaking at 250rpm, for overnight growth. The resulting overnight starter culture is inturn used to inoculate a 3 L baffled flask containing ZYP-5052autoinducing media containing 50 μg/mL of Kanamycin at a dilution of1:1000. Culture volumes ranged from about 600 mL (20% flask volume) toabout 750 mL (25% flask volume). These cultures are grown in a 37° C.incubator shaking at 250 rpm for approximately 5.5 hours and are thentransferred to a 16° C. incubator shaking at 250 rpm for overnightexpression. Cells are harvested by centrifugation (4,000 rpm at 4° C.for 20-30 minutes) and are used immediately, or stored dry at −80° C.until needed.

Example 5 Purification and Quantification of Modified Clostridial Toxins

The following example illustrates methods useful for purification andquantification of any modified Clostridial toxins disclosed in thepresent specification.

For immobilized metal affinity chromatography (IMAC) proteinpurification, E. coli BL21 (DE3) cell pellets used to express a modifiedClostridial toxin, as described in Example 4, are resuspended in ColumnBinding Buffer (25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 2×Protease Inhibitor Cocktail Set III (EMD Biosciences-Calbiochem, SanDiego Calif.); 5 units/mL of Benzonase (EMD Biosciences-Novagen,Madison, Wis.); 0.1% (v/v) Triton-X® 100, 4-octylphenol polyethoxylate;10% (v/v) glycerol), and then are transferred to a cold Oakridgecentrifuge tube. The cell suspension is sonicated on ice (10-12 pulsesof 10 seconds at 40% amplitude with 60 seconds cooling intervals on aBranson Digital Sonifier) in order to lyse the cells and then iscentrifuged (16,000 rpm at 4° C. for 20 minutes) to clarify the lysate.An immobilized metal affinity chromatography column is prepared using a20 mL Econo-Pac column support (Bio-Rad Laboratories, Hercules, Calif.)packed with 2.5-5.0 mL of TALON™ SuperFlow Co²⁺ affinity resin (BDBiosciences-Clontech, Palo Alto, Calif.), which is then equilibrated byrinsing with 5 column volumes of deionized, distilled water, followed by5 column volumes of Column Binding Buffer. The clarified lysate isapplied slowly to the equilibrated column by gravity flow (approximately0.25-0.3 mL/minute). The column is then washed with 5 column volumes ofColumn Wash Buffer (N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 7.8; 500 mM sodium chloride; 10 mM imidazole; 0.1%(v/v) Triton-X® 100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol).The Clostridial toxin is eluted with 20-30 mL of Column Elution Buffer(25 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),pH 7.8; 500 mM sodium chloride; 500 mM imidazole; 0.1% (v/v) Triton-X®100, 4-octylphenol polyethoxylate; 10% (v/v) glycerol) and is collectedin approximately twelve 1 mL fractions. The amount of Clostridial toxincontained in each elution fraction is determined by a Bradford dyeassay. In this procedure, 20 μL aliquots of each 1.0 mL fraction iscombined with 200 μL of Bio-Rad Protein Reagent (Bio-Rad Laboratories,Hercules, Calif.), diluted 1 to 4 with deionized, distilled water, andthen the intensity of the colorimetric signal is measured using aspectrophotometer. The five fractions with the strongest signal areconsidered the elution peak and are combined together. Total proteinyield is determined by estimating the total protein concentration of thepooled peak elution fractions using bovine gamma globulin as a standard(Bio-Rad Laboratories, Hercules, Calif.).

For purification of a modified Clostridial toxin using a FPLC desaltingcolumn, a HiPrep™ 26/10 size exclusion column (Amersham Biosciences,Piscataway, N.J.) is pre-equilibrated with 80 mL of 4° C. Column Buffer(50 mM sodium phosphate, pH 6.5). After the column is equilibrated, aClostridial toxin sample is applied to the size exclusion column with anisocratic mobile phase of 4° C. Column Buffer and at a flow rate of 10mL/minute using a BioLogic DuoFlow chromatography system (Bio-RadLaboratories, Hercules, Calif.). The desalted modified Clostridial toxinsample is collected as a single fraction of approximately 7-12 mL.

For purification of a modified Clostridial toxin using a FPLC ionexchange column, a Clostridial toxin sample that has been desaltedfollowing elution from an IMAC column is applied to a 1 mL Q1™ anionexchange column (Bio-Rad Laboratories, Hercules, Calif.) using aBioLogic DuoFlow chromatography system (Bio-Rad Laboratories, Hercules,Calif.). The sample is applied to the column in 4° C. Column Buffer (50mM sodium phosphate, pH 6.5) and is eluted by linear gradient with 4° C.Elution Buffer (50 mM sodium phosphate, 1 M sodium chloride, pH 6.5) asfollows: step 1, 5.0 mL of 5% Elution Buffer at a flow rate of 1mL/minute; step 2, 20.0 mL of 5-30% Elution Buffer at a flow rate of 1mL/minute; step 3, 2.0 mL of 50% Elution Buffer at a flow rate of 1.0mL/minute; step 4, 4.0 mL of 100% Elution Buffer at a flow rate of 1.0mL/minute; and step 5, 5.0 mL of 0% Elution Buffer at a flow rate of 1.0mL/minute. Elution of Clostridial toxin from the column is monitored at280, 260, and 214 nm, and peaks absorbing above a minimum threshold(0.01 au) at 280 nm are collected. Most of the Clostridial toxin willelute at a sodium chloride concentration of approximately 100 to 200 mM.Average total yields of Clostridial toxin will be determined by aBradford assay.

Expression of a modified Clostridial toxin is analyzed by polyacrylamidegel electrophoresis. Samples purified using the procedure describedabove are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad,Calif.) and are separated by MOPS polyacrylamide gel electrophoresisusing NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels(Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducingconditions. Gels are stained with SYPRO® Ruby (Bio-Rad Laboratories,Hercules, Calif.) and the separated polypeptides are imaged using aFluor-S MAX Multilmager (Bio-Rad Laboratories, Hercules, Calif.) forquantification of Clostridial toxin expression levels. The size andamount of the Clostridial toxin is determined by comparison toMagicMark™ protein molecular weight standards (Invitrogen, Inc,Carlsbad, Calif.).

Expression of modified Clostridial toxin is also analyzed by Westernblot analysis. Protein samples purified using the procedure describedabove are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad,Calif.) and are separated by MOPS polyacrylamide gel electrophoresisusing NuPAGE® Novex 4-12% Bis-Tris precast polyacrylamide gels(Invitrogen, Inc, Carlsbad, Calif.) under denaturing, reducingconditions. Separated polypeptides are transferred from the gel ontopolyvinylidene fluoride (PVDF) membranes (Invitrogen, Inc, Carlsbad,Calif.) by Western blotting using a Trans-Blot® SD semi-dryelectrophoretic transfer cell apparatus (Bio-Rad Laboratories, Hercules,Calif.). PVDF membranes are blocked by incubating at room temperaturefor 2 hours in a solution containing 25 mM Tris-Buffered Saline (25 mM2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl) (pH7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), 0.1%TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovine serumalbumin, 5% nonfat dry milk. Blocked membranes are incubated at 4° C.for overnight in Tris-Buffered Saline TWEEN-20® (25 mM Tris-BufferedSaline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate)containing appropriate primary antibodies as a probe. Primary antibodyprobed blots are washed three times for 15 minutes each time inTris-Buffered Saline TWEEN-20®. Washed membranes are incubated at roomtemperature for 2 hours in Tris-Buffered Saline TWEEN-20® containing anappropriate immunoglobulin G antibody conjugated to horseradishperoxidase as a secondary antibody. Secondary antibody-probed blots arewashed three times for 15 minutes each time in Tris-Buffered SalineTWEEN-20®. Signal detection of the labeled Clostridial toxin arevisualized using the ECL Plus™ Western Blot Detection System (AmershamBiosciences, Piscataway, N.J.) and are imaged with a Typhoon 9410Variable Mode Imager (Amersham Biosciences, Piscataway, N.J.) forquantification of modified Clostridial toxin expression levels.

Example 6 Expression of Modified Clostridial Toxins in a Yeast Cell

The following example illustrates a procedure useful for expressing anyof the modified Clostridial toxins disclosed in the presentspecification in a yeast cell.

To construct a suitable yeast expression construct encoding a modifiedClostridial toxin, restriction endonuclease sites suitable for cloningan operably linked polynucleotide molecule into a pPIC A vector(Invitrogen, Inc, Carlsbad, Calif.) are incorporated into the 5′- and 3′ends of the polynucleotide molecule SEQ ID NO: 136 encodingBoNT/A-ED-PAR1Tb of SEQ ID NO: 85. This polynucleotide molecule issynthesized and a pUCBHB1/BoNT/A-ED-PAR1Tb construct is obtained asdescribed in Example 1. This construct is digested with restrictionenzymes that 1) excise the insert containing the open reading frame ofSEQ ID NO: 136 encoding BoNT/A-ED-PAR1Tb; and 2) enable this insert tobe operably-linked to a pPIC A vector. This insert is subcloned using aT4 DNA ligase procedure into a pPIC A vector that is digested withappropriate restriction endonucleases to yield pPIC A/BoNT/A-ED-PAR1Tb.The ligation mixture is transformed into chemically competent E. coliDH5α cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shockmethod, plated on 1.5% low salt Luria-Bertani agar plates (pH 7.5)containing 25 μg/mL of Zeocin™, and placed in a 37° C. incubator forovernight growth. Bacteria containing expression constructs areidentified as Zeocin™ resistant colonies. Candidate constructs areisolated using an alkaline lysis plasmid mini-preparation procedure andanalyzed by restriction endonuclease digest mapping to determine thepresence and orientation of the insert. This cloning strategy yielded apPIC A expression construct comprising the polynucleotide molecule ofSEQ ID NO: 136 encoding the BoNT/A-ED-PAR1Tb of SEQ ID NO: 85operably-linked to a carboxyl-terminal c-myc and polyhistidine bindingpeptides (FIG. 10).

A similar cloning strategy is used to make pPIC A expression constructscomprising the polynucleotide molecule of SEQ ID NO: 109 encodingBoNT/A-ED-PAR1Tb of SEQ ID NO: 85; SEQ ID NO: 110 or SEQ ID NO: 137encoding BoNT/A-ED-PAR1Xa of SEQ ID NO: 86; the polynucleotide moleculeof SEQ ID NO: 111 or SEQ ID NO: 138 encoding BoNT/A-ED-PAR2Tp of SEQ IDNO: 87; the polynucleotide molecule of SEQ ID NO: 112 or SEQ ID NO: 139encoding BoNT/A-ED-PAR2Xa of SEQ ID NO: 88; the polynucleotide moleculeof SEQ ID NO: 113 or SEQ ID NO: 140 encoding BoNT/A-ED-PAR3Tb of SEQ IDNO: 89; the polynucleotide molecule of SEQ ID NO: 114 or SEQ ID NO: 141encoding BoNT/A-ED-PAR3Xa of SEQ ID NO: 90; the polynucleotide moleculeof SEQ ID NO: 115 or SEQ ID NO: 142 encoding BoNT/A-ED-PAR4Tb of SEQ IDNO: 91; and the polynucleotide molecule of SEQ ID NO: 116 or SEQ ID NO:143 encoding BoNT/A-ED-PAR4Xa of SEQ ID NO: 92.

A similar cloning strategy is used to make pPIC A expression constructscomprising the polynucleotide molecule of SEQ ID NO: 117 encodingBoNT/A-TD-PAR1Xa of SEQ ID NO: 93; SEQ ID NO: 118 or SEQ ID NO: 145encoding BoNT/A-TD-PAR1Xa of SEQ ID NO: 94; the polynucleotide moleculeof SEQ ID NO: 119 or SEQ ID NO: 146 encoding BoNT/A-TD-PAR2Tp of SEQ IDNO: 95; the polynucleotide molecule of SEQ ID NO: 120 or SEQ ID NO: 147encoding BoNT/A-TD-PAR2Xa of SEQ ID NO: 96; the polynucleotide moleculeof SEQ ID NO: 121 or SEQ ID NO: 148 encoding BoNT/A-TD-PAR1Tb of SEQ IDNO: 97; the polynucleotide molecule of SEQ ID NO: 122 or SEQ ID NO: 149encoding BoNT/A-TD-PAR3Xa of SEQ ID NO: 98; the polynucleotide moleculeof SEQ ID NO: 123 or SEQ ID NO: 150 encoding BoNT/A-TD-PAR4Tb of SEQ IDNO: 99; and the polynucleotide molecule of SEQ ID NO: 124 or SEQ ID NO:151 encoding BoNT/A-TD-PAR4Xa of SEQ ID NO: 100.

A similar cloning strategy is used to make pPIC A expression constructscomprising the polynucleotide molecule of SEQ ID NO: 125 encodingBoNT/A-BD-PAR1Tb of SEQ ID NO: 101; the polynucleotide molecule of SEQID NO: 126 or SEQ ID NO: 153 encoding BoNT/A-BD-PAR1Xa of SEQ ID NO:102; the polynucleotide molecule of SEQ ID NO: 127 or SEQ ID NO: 154encoding BoNT/A-BD-PAR2Tp of SEQ ID NO: 103; the polynucleotide moleculeof SEQ ID NO: 128 or SEQ ID NO: 155 encoding BoNT/A-BD-PAR2Xa of SEQ IDNO: 104; the polynucleotide molecule of SEQ ID NO: 129 or SEQ ID NO: 156encoding BoNT/A-BD-PAR3Tb of SEQ ID NO: 105; the polynucleotide moleculeof SEQ ID NO: 130 or SEQ ID NO: 157 encoding BoNT/A-BD-PAR3Xa of SEQ IDNO: 106; the polynucleotide molecule of SEQ ID NO: 131 or SEQ ID NO: 158encoding BoNT/A-BD-PAR4Tb of SEQ ID NO: 107; and the polynucleotidemolecule of SEQ ID NO: 132 or SEQ ID NO: 159 encoding BoNT/A-BD-PAR4Xaof SEQ ID NO: 108.

To construct a yeast cell line expressing a modified Clostridial toxin,pPICZ A/BoNT/A-ED-PAR1Tb is digested with a suitable restrictionendonuclease (i.e., SacI, PmeI or BstXI) and the resulting linearizedexpression construct is transformed into an appropriate P. pastorisMut^(S) strain KM71H using an electroporation method. The transformationmixture is plated on 1.5% YPDS agar plates (pH 7.5) containing 100 μg/mLof Zeocin™ and placed in a 28-30° C. incubator for 1-3 days of growth.Selection of transformants integrating the pPICZ A/BoNT/A-ED-PAR1Tb atthe 5′ AOX1 locus is determined by colony resistance to Zeocin™. Asimilar strategy is used to make a cell line containing a pPICZ Aexpression construct containing SEQ ID NO: 2 used as a control forexpression levels. Cell lines integrating a pPICZ A/BoNT/A-ED-PAR1Tbconstruct is tested for BoNT/A-ED-PAR1Tb expression using a small-scaleexpression test. Isolated colonies from test cell lines that haveintegrated pPICZ A/BoNT/A-ED-PAR1Tb are used to inoculate 1.0 L baffledflasks containing 100 mL of MGYH media and grown at about 28-30° C. in ashaker incubator (250 rpm) until the culture reaches an OD₆₀₀=2-6(approximately 16-18 hours). Cells are harvested by centrifugation(3,000×g at 22° C. for 5 minutes). To induce expression, the cell pelletis resuspended in 15 mL of MMH media and 100% methanol is added to afinal concentration of 0.5%. Cultures are grown at about 28-30° C. in ashaker incubator (250 rpm) for six days. Additional 100% methanol isadded to the culture every 24 hours to a final concentration of 0.5%. A1.0 mL test aliquot is taken from the culture every 24 hours starting attime zero and ending at time 144 hours. Cells are harvested from thealiquots by microcentrifugation to pellet the cells and lysed usingthree freeze-thaw rounds consisting of −80° C. for 5 minutes, then 37°C. for 5 minutes. Lysis samples are added to 2×LDS Sample Buffer(Invitrogen, Inc, Carlsbad, Calif.) and expression from established celllines is measured by Western blot analysis (as described in Example 5)using either anti-BoNT/A, anti-myc or anti-His antibodies in order toidentify lines expressing BoNT/A-ED-PAR1Tb. The P. pastoris Mut^(S)KM71H cell line showing the highest expression level of BoNT/A-ED-PAR1Tbis selected for large-scale expression using commercial fermentationprocedures. Procedures for large-scale expression are as outlined aboveexcept the culture volume is approximately 2.5 L MGYH media grown in a 5L BioFlo 3000 fermentor and concentrations of all reagents will beproportionally increased for this volume.

BoNT/A-ED-PAR1Tb is purified using the IMAC procedure, as described inExample 5. Expression from each culture is evaluated by a Bradford dyeassay, polyacrylamide gel electrophoresis and Western blot analysis (asdescribed in Example 5) in order to determine whether the amounts ofBoNT/A-ED-PAR1Tb produced.

Example 7 Expression of Modified Clostridial Toxins in an Insect Cell

The following example illustrates a procedure useful for expressing anyof the modified Clostridial toxins disclosed in the presentspecification in an insect cell.

To construct suitable an insect expression construct encoding a modifiedClostridial toxin, restriction endonuclease sites suitable for cloningan operably linked polynucleotide molecule into a pBACgus3 vector (EMDBiosciences-Novagen, Madison, Wis.) are incorporated into the 5′- and 3′ends of the polynucleotide molecule SEQ ID NO: 136 encodingBoNT/A-ED-PAR1Tb of SEQ ID NO: 85. This polynucleotide molecule issynthesized and a pUCBHB1/BoNT/A-ED-PAR1Tb construct is obtained asdescribed in Example 1. This construct is digested with restrictionenzymes that 1) excise the insert containing the open reading frame ofSEQ ID NO: 136 encoding BoNT/A-ED-PAR1Tb; and 2) enable this insert tobe operably-linked to a pBACgus3 vector. This insert is subcloned usinga T4 DNA ligase procedure into a pBACgus3 vector that is digested withappropriate restriction endonucleases to yieldpBACgus3/BoNT/A-ED-PAR1Tb. The ligation mixture is transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 100 μg/mL of Ampicillin, and placed in a 37°C. incubator for overnight growth. Bacteria containing expressionconstructs are identified as Ampicillin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pBACgus3 expression construct comprising thepolynucleotide molecule of SEQ ID NO: 136 encoding the BoNT/A-ED-PAR1Tbof SEQ ID NO: 85 operably linked to an amino-terminal gp64 signalpeptide and a carboxyl-terminal, Thrombin cleavable, polyhistidineaffinity binding peptide (FIG. 11).

A similar cloning strategy is used to make pBACgus3 expressionconstructs comprising the polynucleotide molecule of SEQ ID NO: 109encoding BoNT/A-ED-PAR1Tb of SEQ ID NO: 85; SEQ ID NO: 110 or SEQ ID NO:137 encoding BoNT/A-ED-PAR1Xa of SEQ ID NO: 86; the polynucleotidemolecule of SEQ ID NO: 111 or SEQ ID NO: 138 encoding BoNT/A-ED-PAR2Tpof SEQ ID NO: 87; the polynucleotide molecule of SEQ ID NO: 112 or SEQID NO: 139 encoding BoNT/A-ED-PAR2Xa of SEQ ID NO: 88; thepolynucleotide molecule of SEQ ID NO: 113 or SEQ ID NO: 140 encodingBoNT/A-ED-PAR3Tb of SEQ ID NO: 89; the polynucleotide molecule of SEQ IDNO: 114 or SEQ ID NO: 141 encoding BoNT/A-ED-PAR3Xa of SEQ ID NO: 90;the polynucleotide molecule of SEQ ID NO: 115 or SEQ ID NO: 142 encodingBoNT/A-ED-PAR4Tb of SEQ ID NO: 91; and the polynucleotide molecule ofSEQ ID NO: 116 or SEQ ID NO: 143 encoding BoNT/A-ED-PAR4Xa of SEQ ID NO:92.

A similar cloning strategy is used to make pBACgus3 expressionconstructs comprising the polynucleotide molecule of SEQ ID NO: 117encoding BoNT/A-TD-PAR1Xa of SEQ ID NO: 93; SEQ ID NO: 118 or SEQ ID NO:145 encoding BoNT/A-TD-PAR1Xa of SEQ ID NO: 94; the polynucleotidemolecule of SEQ ID NO: 119 or SEQ ID NO: 146 encoding BoNT/A-TD-PAR2Tpof SEQ ID NO: 95; the polynucleotide molecule of SEQ ID NO: 120 or SEQID NO: 147 encoding BoNT/A-TD-PAR2Xa of SEQ ID NO: 96; thepolynucleotide molecule of SEQ ID NO: 121 or SEQ ID NO: 148 encodingBoNT/A-TD-PAR1Tb of SEQ ID NO: 97; the polynucleotide molecule of SEQ IDNO: 122 or SEQ ID NO: 149 encoding BoNT/A-TD-PAR3Xa of SEQ ID NO: 98;the polynucleotide molecule of SEQ ID NO: 123 or SEQ ID NO: 150 encodingBoNT/A-TD-PAR4Tb of SEQ ID NO: 99; and the polynucleotide molecule ofSEQ ID NO: 124 or SEQ ID NO: 151 encoding BoNT/A-TD-PAR4Xa of SEQ ID NO:100.

A similar cloning strategy is used to make pBACgus3 expressionconstructs comprising the polynucleotide molecule of SEQ ID NO: 125encoding BoNT/A-BD-PAR1Tb of SEQ ID NO: 101; the polynucleotide moleculeof SEQ ID NO: 126 or SEQ ID NO: 153 encoding BoNT/A-BD-PAR1Xa of SEQ IDNO: 102; the polynucleotide molecule of SEQ ID NO: 127 or SEQ ID NO: 154encoding BoNT/A-BD-PAR2Tp of SEQ ID NO: 103; the polynucleotide moleculeof SEQ ID NO: 128 or SEQ ID NO: 155 encoding BoNT/A-BD-PAR2Xa of SEQ IDNO: 104; the polynucleotide molecule of SEQ ID NO: 129 or SEQ ID NO: 156encoding BoNT/A-BD-PAR3Tb of SEQ ID NO: 105; the polynucleotide moleculeof SEQ ID NO: 130 or SEQ ID NO: 157 encoding BoNT/A-BD-PAR3Xa of SEQ IDNO: 106; the polynucleotide molecule of SEQ ID NO: 131 or SEQ ID NO: 158encoding BoNT/A-BD-PAR4Tb of SEQ ID NO: 107; and the polynucleotidemolecule of SEQ ID NO: 132 or SEQ ID NO: 159 encoding BoNT/A-BD-PAR4Xaof SEQ ID NO: 108.

To express a modified Clostridial toxin using a baculoviral expressionsystem, about 2.5×10⁶ Sf9 cells are plated in four 60 mm culture dishescontaining 2 mL of BacVector® Insect media (EMD Biosciences-Novagen,Madison, Wis.) and incubated for approximately 20 minutes in a 28° C.incubator. For each transfection, a 50 μL transfection solution isprepared in a 6 mL polystyrene tube by adding 25 μL of BacVector® Insectmedia containing 100 ng of a pBACgus3 construct encoding a modifiedClostridial toxin, such as, e.g., pBACgus3/BoNT/A-ED-PAR1Tb, and 500 ngTlowE transfer plasmid to 25 μL of diluted Insect GeneJuice® containing5 μL Insect GeneJuice® (EMD Biosciences-Novagen, Madison, Wis.) and 20μL nuclease-free water and this solution is incubated for approximately15 minutes. After the 15 minute incubation, add 450 μL BacVector® mediato the transfection solution and mix gently. Using this stocktransfection solution as the 1/10 dilution make additional transfectionsolutions of 1/50, 1/250 and 1/1250 dilutions. Add 100 μL of atransfection solution to the Sf9 cells from one of the four 60 mmculture dishes, twice washed with antibiotic-free, serum-free BacVector®Insect media and incubate at 22° C. After one hour, add 6 mL of 1%BacPlaque agarose-BacVector® Insect media containing 5% bovine serumalbumin. After the agarose is solidified, add 2 mL BacVector® Insectmedia containing 5% bovine serum albumin to the transfected cells andtransfer the cells to a 28° C. incubator for 3-5 days until plaques arevisible. After 3-5 days post-transfection, plaques in the monolayer willbe stained for β-glucuronidase reporter gene activity to test for thepresence of recombinant virus plaques containingpBACgus3/BoNT/A-ED-PAR1Tb by incubating the washed monolayer with 2 mLof BacVector® Insect media containing 30 μL of 20 mg/mL X-Gluc Solution(EMD Biosciences-Novagen, Madison, Wis.) for approximately 2 hours in a28° C. incubator.

After identifying candidate recombinant virus plaques, several candidatevirus plaques are eluted and plaque purified. To elute a recombinantvirus, transfer a plug containing a recombinant virus plaque with asterile Pasteur pipet to 1 mL BacVector® Insect media (EMDBiosciences-Novagen, Madison, Wis.) in a sterile screw-cap vial.Incubate the vial for approximately 2 hours at 22° C. or forapproximately 16 hours at 4° C. For each recombinant virus plaque,2.5×10⁵ Sf9 cells are plated in 35 mm culture dishes containing 2 mL ofBacVector® Insect media (EMD Biosciences-Novagen, Madison, Wis.) andincubated for approximately 20 minutes in a 28° C. incubator. Remove themedia and add 200 μL of eluted recombinant virus. After one hour, add 2mL of 1% BacPlaque agarose-BacVector® Insect media containing 5% bovineserum albumin. After the agarose is solidified, add 1 mL BacVector®Insect media containing 5% bovine serum albumin to the transfected cellsand transfer the cells to a 28° C. incubator for 3-5 days until plaquesare visible. After 3-5 days post-transfection, plaques in the monolayerwill be stained for β-glucuronidase reporter gene activity to test forthe presence of recombinant virus plaques containingpBACgus3/BoNT/A-ED-PAR1Tb by incubating the washed monolayer with 2 mLof BacVector® Insect media containing 30 μL of 20 mg/mL X-Gluc Solution(EMD Biosciences-Novagen, Madison, Wis.) for approximately 2 hours in a28° C. incubator.

To prepare a seed stock of virus, elute a recombinant virus bytransferring a plug containing a recombinant virus plaque with a sterilePasteur pipet to 1 mL BacVector® Insect media (EMD Biosciences-Novagen,Madison, Wis.) in a sterile screw-cap vial. Incubate the vial forapproximately 16 hours at 4° C. Approximately 5×10⁵ Sf9 cells are platedin T-25 flask containing 5 mL of BacVector® Insect media (EMDBiosciences-Novagen, Madison, Wis.) and are incubated for approximately20 minutes in a 28° C. incubator. Remove the media and add 300 μL ofeluted recombinant virus. After one hour, add 5 mL BacVector® Insectmedia containing 5% bovine serum albumin to the transfected cells andtransfer the cells to a 28° C. incubator for 3-5 days until the majorityof cells become unattached and unhealthy. The virus is harvested bytransferring the media to 15 mL snap-cap tubes and centrifuging tubes at1000×g for 5 minutes to remove debris. The clarified supernatant istransferred to fresh 15 mL snap-cap tubes and are stored at 4° C.

To prepare a high titer stock of virus, approximately 2×10⁷ Sf9 cellsare plated in T-75 flask containing 10 mL of BacVector® Insect media(EMD Biosciences-Novagen, Madison, Wis.) and are incubated forapproximately 20 minutes in a 28° C. incubator. Remove the media and add500 μL of virus seed stock. After one hour, add 10 mL BacVector® Insectmedia containing 5% bovine serum albumin to the transfected cells andtransfer the cells to a 28° C. incubator for 3-5 days until the majorityof cells become unattached and unhealthy. The virus is harvested bytransferring the media to 15 mL snap-cap tubes and centrifuging tubes at1000×g for 5 minutes to remove debris. The clarified supernatant istransferred to fresh 15 mL snap-cap tubes and are stored at 4° C. Hightiter virus stocks should contain approximately 2×10⁸ to 3×10⁹ pfu ofbaculovirus.

To express gp64-BoNT/A-ED-PAR1Tb using a baculoviral expression system,about 1.25×10⁸ Sf9 cells are seeded in a 1 L flask containing 250 mL ofBacVector® Insect media and are grown in an orbital shaker (150 rpm) toa cell density of approximately 5×10⁸. The culture is inoculated withapproximately 2.5×10⁹ of high titer stock recombinant baculovirus andincubated for approximately 48 hours in a 28° C. orbital shaker (150rpm). Media is harvested by transferring the media to tubes andcentrifuging tubes at 500×g for 5 minutes to remove debris. Mediasamples are added to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad,Calif.) and expression is measured by Western blot analysis (asdescribed in Example 5) using either anti-BoNT/A or anti-His antibodiesin order to identify baculoviral stocks expressing BoNT/A-ED-PAR1Tb.

BoNT/A-ED-PAR1Tb is purified using the IMAC procedure, as described inExample 5. Expression from each culture is evaluated by a Bradford dyeassay, polyacrylamide gel electrophoresis and Western blot analysis (asdescribed in Example 5) in order to determine whether the amounts ofBoNT/A-ED-PAR1Tb produced.

Example 8 Expression of Modified Clostridial Toxins in a Mammalian Cell

The following example illustrates a procedure useful for expressing anyof the modified Clostridial toxins disclosed in the presentspecification in a mammalian cell.

To construct a suitable mammalian expression construct encoding amodified Clostridial toxin, restriction endonuclease sites suitable forcloning an operably linked polynucleotide molecule into a pSecTag2vector (Invitrogen, Inc, Carlsbad, Calif.) are incorporated into the 5′-and 3′ ends of the polynucleotide molecule SEQ ID NO: 136 encodingBoNT/A-ED-PAR1Tb of SEQ ID NO: 85. This polynucleotide molecule issynthesized and a pUCBHB1/BoNT/A-ED-PAR1Tb construct is obtained asdescribed in Example 1. This construct is digested with restrictionenzymes that 1) excise the insert containing the open reading frame ofSEQ ID NO: 136 encoding BoNT/A-ED-PAR1Tb; and 2) enable this insert tobe operably-linked to a pSecTag2 vector. This insert is subcloned usinga T4 DNA ligase procedure into a pSecTag2 vector that is digested withappropriate restriction endonucleases to yieldpSecTag2/BoNT/A-ED-PAR1Tb. The ligation mixture is transformed intochemically competent E. coli DH5α cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 100 μg/mL of Ampicillin, and placed in a 37°C. incubator for overnight growth. Bacteria containing expressionconstructs are identified as Ampicillin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the insert. This cloningstrategy yielded a pSecTag2 expression construct comprising thepolynucleotide molecule of SEQ ID NO: 136 encoding the BoNT/A-ED-PAR1Tbof SEQ ID NO: 85 operably-linked to a carboxyl-terminal c-myc andpolyhistidine binding peptides (FIG. 12).

A similar cloning strategy is used to make pSecTag2 expressionconstructs comprising the polynucleotide molecule of SEQ ID NO: 109encoding BoNT/A-ED-PAR1Tb of SEQ ID NO: 85; SEQ ID NO: 110 or SEQ ID NO:137 encoding BoNT/A-ED-PAR1Xa of SEQ ID NO: 86; the polynucleotidemolecule of SEQ ID NO: 111 or SEQ ID NO: 138 encoding BoNT/A-ED-PAR2Tpof SEQ ID NO: 87; the polynucleotide molecule of SEQ ID NO: 112 or SEQID NO: 139 encoding BoNT/A-ED-PAR2Xa of SEQ ID NO: 88; thepolynucleotide molecule of SEQ ID NO: 113 or SEQ ID NO: 140 encodingBoNT/A-ED-PAR3Tb of SEQ ID NO: 89; the polynucleotide molecule of SEQ IDNO: 114 or SEQ ID NO: 141 encoding BoNT/A-ED-PAR3Xa of SEQ ID NO: 90;the polynucleotide molecule of SEQ ID NO: 115 or SEQ ID NO: 142 encodingBoNT/A-ED-PAR4Tb of SEQ ID NO: 91; and the polynucleotide molecule ofSEQ ID NO: 116 or SEQ ID NO: 143 encoding BoNT/A-ED-PAR4Xa of SEQ ID NO:92.

A similar cloning strategy is used to make pSecTag2 expressionconstructs comprising the polynucleotide molecule of SEQ ID NO: 117encoding BoNT/A-TD-PAR1Xa of SEQ ID NO: 93; SEQ ID NO: 118 or SEQ ID NO:145 encoding BoNT/A-TD-PAR1Xa of SEQ ID NO: 94; the polynucleotidemolecule of SEQ ID NO: 119 or SEQ ID NO: 146 encoding BoNT/A-TD-PAR2Tpof SEQ ID NO: 95; the polynucleotide molecule of SEQ ID NO: 120 or SEQID NO: 147 encoding BoNT/A-TD-PAR2Xa of SEQ ID NO: 96; thepolynucleotide molecule of SEQ ID NO: 121 or SEQ ID NO: 148 encodingBoNT/A-TD-PAR1Tb of SEQ ID NO: 97; the polynucleotide molecule of SEQ IDNO: 122 or SEQ ID NO: 149 encoding BoNT/A-TD-PAR3Xa of SEQ ID NO: 98;the polynucleotide molecule of SEQ ID NO: 123 or SEQ ID NO: 150 encodingBoNT/A-TD-PAR4Tb of SEQ ID NO: 99; and the polynucleotide molecule ofSEQ ID NO: 124 or SEQ ID NO: 151 encoding BoNT/A-TD-PAR4Xa of SEQ ID NO:100.

A similar cloning strategy is used to make pSecTag2 expressionconstructs comprising the polynucleotide molecule of SEQ ID NO: 125encoding BoNT/A-BD-PAR1Tb of SEQ ID NO: 101; the polynucleotide moleculeof SEQ ID NO: 126 or SEQ ID NO: 153 encoding BoNT/A-BD-PAR1Xa of SEQ IDNO: 102; the polynucleotide molecule of SEQ ID NO: 127 or SEQ ID NO: 154encoding BoNT/A-BD-PAR2Tp of SEQ ID NO: 103; the polynucleotide moleculeof SEQ ID NO: 128 or SEQ ID NO: 155 encoding BoNT/A-BD-PAR2Xa of SEQ IDNO: 104; the polynucleotide molecule of SEQ ID NO: 129 or SEQ ID NO: 156encoding BoNT/A-BD-PAR3Tb of SEQ ID NO: 105; the polynucleotide moleculeof SEQ ID NO: 130 or SEQ ID NO: 157 encoding BoNT/A-BD-PAR3Xa of SEQ IDNO: 106; the polynucleotide molecule of SEQ ID NO: 131 or SEQ ID NO: 158encoding BoNT/A-BD-PAR4Tb of SEQ ID NO: 107; and the polynucleotidemolecule of SEQ ID NO: 132 or SEQ ID NO: 159 encoding BoNT/A-BD-PAR4Xaof SEQ ID NO: 108.

To transiently express modified Clostridial toxin in a cell line, about1.5×10⁵ SH-SY5Y cells are plated in a 35 mm tissue culture dishcontaining 3 mL of complete Dulbecco's Modified Eagle Media (DMEM),supplemented with 10% fetal bovine serum (FBS), 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilcells reach a density of about 5×10⁵ cells/ml (6-16 hours). A 500 μLtransfection solution is prepared by adding 250 μL of OPTI-MEM ReducedSerum Medium containing 15 μL of LipofectAmine 2000 (Invitrogen,Carlsbad, Calif.) incubated at room temperature for 5 minutes to 250 μLof OPTI-MEM Reduced Serum Medium containing 5 μg of a pSecTag2expression construct encoding a modified Clostridial toxin, such as,e.g., pSecTag2/BoNT/A-ED-PAR1Tb. This transfection is incubated at roomtemperature for approximately 20 minutes. The complete, supplementedDMEM media is replaced with 2 mL of OPTI-MEM Reduced Serum Medium andthe 500 μL transfection solution is added to the SH-SY5Y cells and thecells are incubated in a 37° C. incubator under 5% carbon dioxide forapproximately 6 to 18 hours. Transfection media is replaced with 3 mL offresh complete, supplemented DMEM and the cells are incubated in a 37°C. incubator under 5% carbon dioxide for 48 hours. Both media and cellsare collected for expression analysis of BoNT/A-ED-PAR1Tb. Media isharvested by transferring the media to 15 mL snap-cap tubes andcentrifuging tubes at 500×g for 5 minutes to remove debris. Cells areharvested by rinsing cells once with 3.0 mL of 100 mM phosphate-bufferedsaline, pH 7.4 and lysing cells with a buffer containing 62.6 mM2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl), pH6.8 and 2% sodium lauryl sulfate (SDS). Both media and cell samples areadded to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad, Calif.) andexpression is measured by Western blot analysis (as described in Example5) using either anti-BoNT/A, anti-c-myc or anti-His antibodies in orderto identify pSecTag2 constructs expressing BoNT/A-ED-PAR1Tb. A similarprocedure can be used to transiently express a pSecTag2 constructencoding any of the modified Clostridial toxin of SEQ ID NO: 86 to SEQID NO: 108.

To generate a stably-integrated cell line expressing a modifiedClostridial toxin, approximately 1.5×10⁵ SH-SY5Y cells are plated in a35 mm tissue culture dish containing 3 mL of complete DMEM, supplementedwith 10% FBS, 1× penicillin/streptomycin solution (Invitrogen, Inc,Carlsbad, Calif.) and 1×MEM non-essential amino acids solution(Invitrogen, Inc, Carlsbad, Calif.), and grown in a 37° C. incubatorunder 5% carbon dioxide until cells reach a density of about 5×10⁵cells/ml (6-16 hours). A 500 μL transfection solution is prepared byadding 250 μL of OPTI-MEM Reduced Serum Medium containing 15 μL ofLipofectAmine 2000 (Invitrogen, Carlsbad, Calif.) incubated at roomtemperature for 5 minutes to 250 μL of OPTI-MEM Reduced Serum Mediumcontaining 5 μg of a pSecTag2 expression construct encoding a modifiedClostridial toxin, such as, e.g., pSecTag2/BoNT/A-ED-PAR1Tb. Thistransfection solution is incubated at room temperature for approximately20 minutes. The complete, supplemented DMEM media is replaced with 2 mLof OPTI-MEM Reduced Serum Medium and the 500 μL transfection solution isadded to the SH-SY5Y cells and the cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 6 to 18 hours.Transfection media is replaced with 3 mL of fresh complete, supplementedDMEM and cells are incubated in a 37° C. incubator under 5% carbondioxide for approximately 48 hours. Media is replaced with 3 mL of freshcomplete DMEM, containing approximately 5 μg/mL of Zeocin™, 10% FBS, 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.). Cells are incubated in a 37° C. incubator under 5% carbondioxide for approximately 3-4 weeks, with old media being replaced withfresh Zeocin™-selective, complete, supplemented DMEM every 4 to 5 days.Once Zeocin™-resistant colonies are established, resistant clones arereplated to new 35 mm culture plates containing fresh complete DMEM,supplemented with approximately 5 μg/mL of Zeocin™, 10% FBS, 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), until these cells reach a density of 6 to 20×10⁵ cells/mL. Totest for expression of BoNT/A-ED-PAR1Tb from SH-SY5Y cell lines thathave stably-integrated a pSecTag2/BoNT/A-ED-PAR1Tb, approximately1.5×10⁵ SH-SY5Y cells from each cell line are plated in a 35 mm tissueculture dish containing 3 mL of Zeocin™-selective, complete,supplemented DMEM and grown in a 37° C. incubator under 5% carbondioxide until cells reach a density of about 5×10⁵ cells/ml (6-16hours). Media is replaced with 3 mL of fresh Zeocin™-selective,complete, supplemented DMEM and cells are incubated in a 37° C.incubator under 5% carbon dioxide for 48 hours. Both media and cells arecollected for expression analysis of BoNT/A-c-myc-His. Media isharvested by transferring the media to 15 mL snap-cap tubes andcentrifuging tubes at 500×g for 5 minutes to remove debris. Cells areharvest by rinsing cells once with 3.0 mL of 100 mM phosphate-bufferedsaline, pH 7.4 and lysing cells with a buffer containing 62.6 mM2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl), pH6.8 and 2% sodium lauryl sulfate (SDS). Both media and cell samples areadded to 2×LDS Sample Buffer (Invitrogen, Inc, Carlsbad, Calif.) andexpression is measured by Western blot analysis (as described in Example5) using either anti-BoNT/A, anti-c-myc or anti-His antibodies in orderto identify SH-SY5Y cell lines expressing BoNT/A-ED-PAR1Tb. Theestablished SH-SY5Y cell line showing the highest expression level ofBoNT/A-ED-PAR1Tb is selected for large-scale expression using 3 Lflasks. Procedures for large-scale expression are as outlined aboveexcept the starting volume is approximately 800-1000 mL of complete DMEMand concentrations of all reagents are proportionally increased for thisvolume. A similar procedure can be used to stably express a pSecTag2construct encoding any of the modified Clostridial toxin of SEQ ID NO:86 to SEQ ID NO: 108.

BoNT/A-ED-PAR1Tb is purified using the IMAC procedure, as described inExample 5. Expression from each culture is evaluated by a Bradford dyeassay, polyacrylamide gel electrophoresis and Western blot analysis (asdescribed in Example 5) in order to determine whether the amounts ofBoNT/A-ED-PAR1Tb produced.

Although aspects of the present invention have been described withreference to the disclosed embodiments, one skilled in the art willreadily appreciate that the specific examples disclosed are onlyillustrative of these aspects and in no way limit the present invention.Various modifications can be made without departing from the spirit ofthe present invention.

1. A method of treating a human in need thereof comprisingadministration of a therapeutically effective amount of modifiedClostridial toxin, said modified Clostridial toxin comprising: a) aprotease-activated G protein-coupled receptor (PAR) ligand domain,wherein the PAR ligand domain comprises a masked or unmasked PAR liganddomain, and wherein the unmasked PAR ligand domain binds to a PAR ligandbinding domain of a target cell under physiological conditions; b) aClostridial toxin enzymatic domain that proteolytically cleaves asubstrate of a Clostridial toxin; c) a Clostridial toxin translocationdomain that executes a translocation step of a Clostridial toxinintoxication process; and d) a Clostridial toxin binding domain thatthat executes a cell binding step of a Clostridial toxin intoxicationprocess.
 2. The method according to claim 1, wherein the PAR liganddomain is operationally-linked to the amino terminus of the Clostridialtoxin enzymatic domain.
 3. The method according to claim 2, wherein themodified Clostridial toxin comprises an amino to carboxyl singlepolypeptide linear order comprising the PAR ligand domain, theClostridial toxin enzymatic domain, the Clostridial toxin translocationdomain and the Clostridial toxin binding domain.
 4. The method accordingto claim 2, wherein the modified Clostridial toxin comprises an amino tocarboxyl single polypeptide linear order comprising the PAR liganddomain, the Clostridial toxin enzymatic domain, the Clostridial toxinbinding domain and the Clostridial toxin translocation domain.
 5. Themethod according to claim 1, wherein the PAR ligand domain isoperationally-linked to the amino terminus of the Clostridial toxintranslocation domain.
 6. The method according to claim 5, wherein themodified Clostridial toxin comprises an amino to carboxyl singlepolypeptide linear order comprising the Clostridial toxin bindingdomain, the Clostridial toxin enzymatic domain, the PAR ligand domainand the Clostridial toxin translocation domain.
 7. The method accordingto claim 5, wherein the modified Clostridial toxin comprises an amino tocarboxyl single polypeptide linear order comprising the Clostridialtoxin enzymatic domain, the PAR ligand domain, the Clostridial toxintranslocation domain and the Clostridial toxin binding domain.
 8. Themethod according to claim 1, wherein the PAR ligand domain isoperationally-linked to the amino terminus of the Clostridial toxinbinding domain.
 9. The method according to claim 8, wherein the modifiedClostridial toxin comprises an amino to carboxyl single polypeptidelinear order comprising the Clostridial toxin enzymatic domain, the PARligand domain, the Clostridial toxin binding domain and the Clostridialtoxin translocation domain.
 10. The method according to claim 1, whereinthe modified Clostridial toxin further comprises a protease cleavagesite in the masked PAR ligand domain; and wherein cleavage of theprotease cleavage site unmasks the PAR ligand domain.
 11. The methodaccording to claim 1, wherein the PAR ligand domain comprises a PAR1ligand domain.
 12. The method according to claim 11, wherein the PAR1ligand Domain comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO:
 133. 13. Themethod according to claim 1, wherein the modified Clostridial toxin is amodified Botulinum toxin comprising a PAR ligand domain, a Botulinumtoxin enzymatic domain, a Botulinum toxin translocation domain and aBotulinum toxin binding domain.
 14. The method according to claim 13,wherein the modified Botulinum toxin is a modified BoNT/A comprising aPAR ligand domain, a BoNT/A enzymatic domain, a BoNT/A translocationdomain and a BoNT/A binding domain.
 15. The method of claim 1, whereinthe human is treated for achalasia, adult spasticity, anal fissure, backpain, blepharospasm, bruxism, cervical dystonia, essential tremor,glabellar lines or hyperkinetic facial lines, headache, hemifacialspasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral palsy,multiple sclerosis, myoclonic disorders, nasal labial lines, spasmodicdysphonia, strabismus and/or VII nerve disorder.